Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody / 中国生物制品学杂志

@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins / 中国生物制品学杂志

@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Eukaryotic expression and biological activity identification of fusion protein ABD-Fc-IL-2 / 中国生物制品学杂志

@#Objective To express the fusion protein ABD-Fc-IL-2 in eukaryotic cells and detect its biological activity. Methods The target gene SP-ABD-Fc-IL-2 was amplified by direct and overlapping PCR,and then ligated to vector pcDNA3. 1(+).The obtained recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was transiently transfected into CHO-S cells to express the fusion protein ABD-Fc-IL-2,which was purified by Protein A beads affinity chromatography. The specificity of the purified fusion protein was detected by Western blot,the biological activity was detected by CTLL-2/MTT cell proliferation colourimetry,and the interaction between ABD fragment and human serum albumin(HSA)was detected by pull down/Western blot. Results The recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was constructed correctly as identified by restriction analysis and sequencing. The purified fusion protein ABD-Fc-IL-2 showed a purity of 90% and bound specifically to mouse anti-IL-2 monoclonal antibody with the biological activity of 3. 29 × 108IU/mL. The ABD of fusion protein and HSA bound to each other. Conclusion The eukaryotic fusion protein ABD-Fc-IL-2 had high biological activity,which promoted the proliferation of CTLL-2 cells and maintained the binding ability of ABD fragment to HSA.

Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Therapeutic effects of IL-6 and BAFF fusion protein eukaryotic expression plasmid on animal model of Sj?gren′s syndrome / 中华微生物学和免疫学杂志

Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.

Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27 / 西安交通大学学报(医学版)

【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.

DNA Damage-Inducible Transcript 4 (Ddit4) Overexpression Could Inhibit Proliferation and Promote Apoptosis of HT-22 Cells through the Wnt / β-catenin Signaling Pathway / 中国生物化学与分子生物学报

Neural tube defects (NTDs) are congenital defect diseases caused by cell proliferation and apoptosis disorders. Using RNA-Seq assays, we found the increased expression of DNA damage-inducible transcript 4 (Ddit4) in embryonic brain tissues from NTD fetuses. In this study, we intend to explore the effects of Ddit4 overexpression on the proliferation and apoptosis of HT-22 cells and related mechanisms to lay the foundation for the study of the role of Ddit4 in NTDs. According to the mouse Ddit4 sequence, we constructed the eukaryotic expression vector pEX-3-Ddit4. The results of restriction enzyme analysis and sequencing showed that the eukaryotic expression vector pEX-3-Ddit4 was successfully constructed. qRT-PCR and Western blotting results showed that the expression level of Ddit4 in HT-22 cells was significantly increased after transfection of PEX-3-Ddit4 (P < 0. 01) . CCK-8 and Western blotting results showed that Ddit4 overexpression decreased the proliferation of HT-22 cells (P < 0. 01) . Flow cytometry showed that Ddit4 overexpression increased the proportion of cells in the G

Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein / 解剖学报

Objective To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein (pEGFP)-N1-CPNE3, and identify the expression and localization of Copine-3 protein in cells. Methods The Copine-3 coding sequences (CPNE3) was amplified by RT-PCR from human bronchial epithelial (HBE) cells and inserted into eukaryotic expression vector pEGFP-Nl. The recombinant plasmid pEGFP-Nl-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T and H1299 cells was detected by Western blotting analysis. Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry. Results CPNE3 was successfully constructed into the eukaryotic expression vector pEGFP-Nl and expressed in 293T and H1299 cells. Furthermore, the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining, immuno Western blotting and immunohistochemistry in those cells and clinical samples. Conclusion The eukaryotic expression vector pEGFP-Nl-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.

Construction of eukaryotic expression vector and bioinformatics analysis of human kidney and brain protein (KIBRA) / 西安交通大学学报(医学版)

【Objective】 To clone the full-length of human kidney and brain protein (KIBRA) coding sequence in eukaryotic expression vector and provide a model for studying the biological function of KIBRA in breast cancer cells. 【Methods】 Total RNA of human breast cancer cell line MCF7 was extracted. After reverse transcription, the full length of KIBRA (NM_001161661.2) coding region was amplified by PCR, and cloned into eukaryotic expression vector pCMV-Blank. After identification, it was defined officially as pCMV-KIBRA. Then it was transfected into MCF7 cells, and the expression of KIBRA was detected by real-time PCR and Western blotting after 48 hours. The primary, secondary and tertiary structures and post-transcriptional modification sites of KIBRA were analyzed with bioinformatics software. 【Results】 Bacterial PCR, double enzyme digestion and DNA sequencing results showed that the correct sequence of KIBRA was inserted into the vector pCMV-KIBRA. The mRNA and protein expressions of KIBRA were significantly increased in MCF7 cells transfected with pCMV-KIBRA. Bioinformatics analysis showed that KIBRA was composed of 1119 amino acids. There were 52 phosphorylation sites, 1 acetylation site and 5 ubiquitination sites, and the protein structure was mainly α-helix and random coil. 【Conclusion】 The eukaryotic expression vector of full-length of human KIBRA coding sequence was successfully constructed and overexpressed in breast cancer cell line MCF7, which can lay a foundation for studying the biological function of KIBRA in breast cancer.

Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

Gene cloning, protein expression and examination of biological activity of chicken CD40L / 生物工程学报

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.

Eukaryotic expression, protein purification and biological effects research of human CS1-Fc fusion protein / 生物工程学报

Signaling lymphocyte activation family 7 (SLAMF7/CS1) is a cell surface glycoprotein that is highly expressed in multiple myeloma cells. CS1 is a sensitive and specific biomarker for multiple myeloma. CAR-T cell immunotherapy is a new method for the treatment of multiple myeloma. CS1 CAR-T cell immunotherapy has good effect on relapsed refractory multiple myeloma. To detect the expression efficiency of CS1 CAR on CS1 CAR-T cells and to find an auxiliary means to CAR-T cell immunotherapy, we prepared a CS1-Fc fusion protein. First, the extracellular domain of CS1 was amplified from the existing plasmid by PCR and ligated with human IgG1-Fc fragment by overlap extension PCR. The recombinant fragment was ligated into pMH3 eukaryotic expression vector. After restriction enzyme digestion and DNA sequencing, the pMH3-CS1-Fc-his recombinant plasmid was successfully constructed. The recombinant plasmid was transfected into Chinese hamster ovary cell (CHO-S) by liposome. The expression of the CS1-Fc fusion protein in CHO-S cells was identified by flow cytometry after G418 pressure screening. Next, the CS1-Fc fusion protein was purified by nickel column. Western-blot analysis showed that molecular weight of the fusion protein was about 70 kDa was identified by Western blotting. The CS1-Fc fusion protein couldeffectively detect the expression rate of CS1 CAR and promote the activation, proliferation andcytokines secretion of the CS1 CAR-T cells. The results will lay the experimental foundation for the in vitro detection and potentiation of CAR-T cells in multiple myeloma treated with CS1 CAR-T cell.

Construction of eukaryotic expression vector of hypoxic inducible VEGF and its in vitro delivery application and functional identification / 解放军医学杂志

Objective To construct the eukaryotic expression vector of hypoxia inducible vascular endothelial growth factor (VEGF), and establish its in vitro delivery method. Methods Erythropoietin (EPO) enhancer was inserted into eukaryotic expression vector pGL4.73 [hRluc/SV40] (pSV) promoter by gene recombination technique to construct hypoxia inducible expression system (pEPO-SV). Renilla luciferase (Rluc) was used as downstream reporter gene. Then the VEGF165 gene was inserted into the pEPO-SV plasmid instead of Rluc, and the pEPO-SV-VEGF and pSV-VEGF expression vectors were obtained by inserting the pEPO-SV-VEGF gene into pSV as control. The pSV plasmid expressing Rluc or VEGF165 and pEPO-SV plasmid were transfected in vitro into human embryonic kidney 293T cells. The expression of Rluc or VEGF165 was used to identify the hypoxia induction function of the constructed vector after being treated under normal and hypoxic conditions for 24h and 48h. The intracellular delivery method of plasmids was then established based on poly (lactic acid-glycolic acid) copolymer (PLGA) nanoparticles as carrier, and the efficiency of the eukaryotic expression plasmids induced by hypoxia was evaluated under the in vitro hypoxia model. Results In the construction of plasmid, the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction endonuclease digestion, PCR amplification and DNA sequencing. The plasmid expressing Rluc or VEGF165 was transfected into 293T cells respectively. There was no significant difference in the expression of reporter gene Rluc (one, plasmid pSV and pEPO-SV fluorescence expression values were 2448.24±158.51 and 3173.97±379.92, the second, plasmid pSV and pEPO-SV fluorescence expression values were 55 500.00±3237.05 and 51 193.18±866.32, respectively) or target gene VEGF165 in normal culture (P>0.05). But the expression of Rluc (In the cobalt chloride of hypoxia, the fluorescence expression values of pSV and pEPO-SV were 4857.70±1223.28 and 16 432.64±1618.73, respectively. In the hypoxia incubator, the fluorescence expression values of pSV and pEPO-SV were 2504.45±213.20 and 17 274.35±685.60, respectively) or VEGF165 in hypoxia was significantly higher than that in control group (P<0.01). The results showed that the constructed pEPO-SV and pEPO-SV-VEGF plasmids had typical hypoxia inducible expression activity. PLGA nanoparticles were used to in vitro deliver pEPO-SV and pSV in 293T cells. The results of detecting the reporter gene Rluc in normal culture and hypoxic conditions were consistent with those mentioned above, that is, under normal conditions, the 24h and 48h fluorescence expression values of plasmids pSV and pEPO-SV were 149.44±4.01 and 127.09±15.05, 1074.91±114.78 and 1064.56±137.48, respectively; under hypoxic conditions, the 24h and 48h fluorescence expression values of pSV and pEPO-SV were 3265.34±440.00 and 8828.87±637.03, 3202.06±33.43 and 9114.75±292.06, respectively. Conclusion A typical hypoxia inducible VEGF eukaryotic expression system has been successfully established, and an in vitro effective delivery method is also established, which may have an important application prospect in ischemia, hypoxia and other tissue injury diseases.

Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2 / 生物工程学报

The aim of this study is to prepare monoclonal anti-human Lp-PLA2 antibodies, and establish a rapid and accurate immunochromatographic Lp-PLA2 assay used in community medical institution. The gene sequence of human Lp-PLA2 was obtained from NCBI to construct the expression plasmid. Lp-PLA2 protein expressed in CHO-K1 cells was used to immune BALB/c mice. The monoclonal antibodies were produced in mouse ascites after hybridoma cells screening. Antibodies were evaluated by SDS-PAGE, ELISA and other methods. The Lp-PLA2 test strip was prepared based on sandwich method and evaluated with the portable detection instrument. The affinity of the paired antibodies, PLA1 and PLA5, both reached 1×10⁻⁸. The antibody subclass was IgG1. Both antibodies recognized the Lp-PLA2 protein in the blood specifically. The Lp-PLA2 test strip was prepared based on sandwich method, with linear range of 20-2000 ng/mL. The Lp-PLA2 test strip correlated well with the diaDexus ELISA test kit. In conclusion, the paired antibodies were successfully prepared with high affinity and specificity. The immunochromatographic test of Lp-PLA2 provided a fast and accurate method to detect the concentration of Lp-PLA2 in blood sample for clinical use in the community medical institution and could contribute to the management of cardiovascular diseases.

Eukaryotic expression and serodiagnostic potential of HIV-1 p24 antigen / 中华微生物学和免疫学杂志

Objective To express HIV-1 capsid p24 antigen in an eukaryotic expression system and to evaluate its antigenicity and potential in the early diagnosis of HIV. Methods The full-length gene of HIV-1 p24 and the signal peptide DRVI gene were amplified by PCR. The signal peptide DRVI preceding the p24 gene was introduced using fusion PCR, and cloned into vector pDRVI1. 0. Two recombinant plas-mids pDRVI-p24 and pDRVI-p24s were constructed and transfected into 293F cells. Expression and secre-tion of p24 protein were detected by SDS-PAGE, Ni-NTA column chromatography and molecular sieve were used to purify p24s protein. The purified protein was identified by Western blot and indirect ELISA using hu-man/mouse HIV-1-positive serum samples. Results The eukaryotic expression system for HIV-1 p24 anti-gen was successfully established with high efficiency. The target protein of interest with the signal peptide DRVI was obviously detected in the supernatants of cell culture. The recombinant protein had good specifici-ty and sensitivity based on the results of serological tests using serum samples of five HIV-1-positive and five HIV-negative mice , 30 HIV-1-positive patients and 50 HIV-1-negative healthy individuals . Conclusions The eukaryotic expression system for HIV-1 p24 antigen was successfully established. The purified HIV-1 p24s antigen had good antigenicity. An indirect ELISA assay with good specificity and sensitivity for the de-tection of HIV-1 was preliminarily constructed and showed great potential for application.

Establishment of EA-D-IgA antibody detection method and its diagnostic efficacy for nasopharyngeal carcinoma in different clinical stage / 肿瘤研究与临床

Objective To establish a serological detection method for EA-D-IgA antibody,and to evaluate its diagnostic efficacy for nasopharyngeal carcinoma in different clinical stage.Methods EA-D-IgA antibody serological detection method was established by using the polypropylene microplate with eukaryotic expression product of BMRF1 whole gene fragment of EB virus.Fifteen early stage (stage Ⅰ and Ⅱ) and 48 advanced (stage Ⅲ and Ⅳ) patients with nasopharyngeal carcinoma in Shanxi Provincial Cancer Hospital and Shanxi Dayi Hospital from April 2012 to August 2017,and serum samples from 40 patients with rhinitis who were treated at Shanxi Dayi Hospital from October 2016 to October 2017 were examined respectively by using the constructed EA-D-IgA antibody detection method.The positive detection rate of EA-D-IgA antibodies in different groups was calculated.When the patients with rhinitis were used as the differential control,the diagnostic efficacy of this index for different stages of nasopharyngeal carcinoma was evaluated.Results EA-D-IgA antibody serological method was successfully established.The positive detection rate of EA-D-IgA antibody in early nasopharyngeal carcinoma,advanced nasopharyngeal carcinoma and rhinitis control was 60.0 ％ (10/15),68.3 ％ (33/48) and 5.0 ％ (2/40) respectively.The differences between early stage nasopharyngeal carcinoma and the rhinitis control,advanced nasopharyngeal carcinoma and the rhinitis control were statistically significant (x2 =20.625,P =0.000;x2 =37.017,P =0.000).The difference between early nasopharyngeal carcinoma and advanced nasopharyngeal carcinoma was not statistically significant (x2 =0.394,P =0.530).When compared with the patients with rhinitis,the diagnostic sensitivity,specificity,positive predictive value,negative predictive value was 60.0 ％ and 68.3 ％,95.0 ％ and 95.0 ％,81.8 ％ and 94.3 ％,86.4 ％ and 71.7 ％ respectively in early nasopharyngeal carcinoma and advanced nasopharyngeal carcinoma.Conclusion The method constructed in this study effectively improves the efficacy of EA-D-IgA antibody detection in serological diagnosis of nasopharyngeal carcinoma,which can be used as an adjunct for early diagnosis of nasopharyngeal carcinoma,yet not as a reference for clinical staging.

Construction and characterization of a eukaryotic expression vector of full-length and mature IL-37b / 中国比较医学杂志

Objective To construct a eukaryotic expression vector pEGFP N1/IL-37b of full-length and mature IL-37b,and to detect the expression of both full-length and mature IL-37b in RAW 264.7 cells, a mouse macrophage cell line. Methods To construct the eukaryotic vectors of full-length and mature IL-37b by using plasmid pUBC/IL-37b as a template containing the coding region of IL-37b full-length gene. To detect the expression of IL-37b by western blot and confocal microscopy after transfected the recombinant plasmid into RAW 264.7 cells, and to detect the inhibition of full-length and mature IL-37b on IL-6 production by real-time PCR. Results Eukaryotic vectors pEGFP N1/IL-37b expressed full-length and mature IL-37b after transfection in cells, which inhibited LPS-induced IL-6 production. Conclusions Eukaryotic vectors of full-length and mature IL-37b can be successfully constructed,and lays a foundation for further study of anti-inflammation functions and mechanisms of IL-37b.

Lactate dehydrogenase A promotes the growth of human melanoma cell line MUM-2B / 眼科新进展

Objective To construct the eukaryotic expression vector of LDHA with Flag label and detect its effects on the growth of human choroidal melanoma (CM) MUM-2B cells.Methods CM cells line MUM-2B subcultured by the Military Academy of Sciences were divided into two groups:experimental group and control group.The experimental group was transiently transfected with Flag-LDHA plasmid,and the control group was transiently transfected with Flag plasmid.Using the Flag-LDHA with GST label as a template,the LDHA gene was amplified by polymerase chain reaction (PCR),which then was inserted into eukaryotic expression vector of Flag,and the recombinant plasmid Flag-LDHA was identified by bacterial liquid PCR,double enzyme digestion and sequencing,both which were transiently transfected into human CM MUM-2B cells after successful identification,and finally,its expression was determined by Western blot.The biology behaviors of melanoma cell line MUM-2B transfected with Flag-LDHA and Flag plasmid were analyzed by counting Kit-8 (CCK8) assays.Results The coding region sequence of LDHA gene of approximately 1000 bp was harvested from PCR amplification,which was successfully cloned into the Flag vector.Compared with the control group,the PCR result of the bacterial liquid in the experimental group was positive.The double enzyme digestion results showed that eukaryotic expression vector of Flag with a length of about 4000 bp Flag vector and a 1000 bp LDHA gene band.And the sequencing results indicated that the inserted sequence was completely in consonance with the coding sequence of the LDHA gene.Western blot results showed the successful expression of recombinant plasmid Flag-LDHA in MUM-2B melanoma cells.CCK8 assays demonstrated that Flag-LDHA recombinant plasmid could promote the growth of melanoma cell line MUM-2B.Conclusion The eukaryotic expression vector of Flag-LDHA was successfully constructed,which can promote the growth of melanoma cell line MUM-2B.This will lay the foundation for studying the function of LDHA in the initiation and progression of human CM.