Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC / 生物工程学报

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

Cloning,expression and biological activity of m6A binding protein YTHDF2 / 军事医学

Objective To construct the recombinant plasmid of YTH domain family 2(YTHDF2)and express it in E.coli in order to obtain YTHDF2 fusion protein that was capable of binding m 6A-modified RNA.Methods The coding region of YTHDF2 gene was amplified by RT-PCR.The recombinant plasmid pET-28a-YTHDF2 was constructed and expressed in E.coli.The fusion protein was purified by Ni2+-NTA resin affinity chromatography, while the fusion protein activity was analyzed by Ni2+-NTA magnetic spheres.Results and Conclusion The recombinant YTHDF2 protein was expressed in E.coli BL21(DE3)and purified.YTHDF2 fusion protein was capable of binding RNA with m 6A-modification. The preparation of YTHDF2 fusion protein provides an essential tool to study the biological function of RNA with m6A-modification.

Construction and expression of cell-penetrating peptide CCL fusion protein expression vector / 中国感染控制杂志

Objective To evaluate the construction of expression vector for fusion protein of cell-penetrating pep-tide CCL (PEP-CCL).Methods CCL6-PEP-6XHis was inserted into plasmid pABP,pABP-CCL6-PEP plasmid was extracted and then transfected into HEK293 cells,CCL6-PEP-6XHis was expressed and purified by chromatog-raphy and detected with Western Blot.Results PEP-CCL express vector was successfully constructed and purified. PCR product of CCL6-PEP-6XHis Tag was ligated with T vector,recombinant was transferred into the host cells, then host cells were cultured,plasmid was extracted and sequenced,the sequence was identical to targeted gene. CCL6-PEP-6XHis was successfully inserted into the eukaryotic expression vector pABP,plasmid was extracted and digested,electrophoresis results revealed that a fragment with 430bp was digested by Hind Ⅲ+XbaⅠ,which was identical to the expected value.Western Blot revealed that CCL6-PEP fusion protein could be recognized by His monoclonal antibody.Conclusion PEP-CCL express vector can be constructed and expressed in eukaryotic cells.

Inhibitory effect of tumor growth of recombinant protein fused with cardiac troponin I and artificial peptide / 中国药理学通报

Aim To examine the inhibitory effect of re-combinant cardiac troponin fusion protein composed of subunit I and artificial peptide which was called CIS on tumor growth. Methods The CIS ’ s effect on the growth of human umbilical vein endothelial cells ( HU-VEC) was examined using MTT assay in vitro. Chick chorioallantoic membrane model was applied to study the alteration of angiogenesis treated with purified re-combinant CIS protein. The effect of tumor growth trea-ted with CIS was observed using several in vivo mice xenograft models. Results There was a statistically significant reduction in HUVEC cell proliferative rate when the cells were treated with purified CIS fusion protein, which was also shown in a dose-dependent manner. A decreased amount of new blood vessel for-mation ( angiogenesis) on chick embryo chorioallantoic membranes was observed in recombinant CIS protein treated group compared to the untreated control group. A significant inhibition of tumor growth rate was a-chieved in CIS treated mice compared to CIS untreated control mice in 6 different mouse xenograft models. Conclusions The fusion protein CIS shows the inhibi-tory effect on the tumor growth in our in vivo mouse models, and such inhibition could be mediated by the mechanism of CIS’ s effect on the decrease of HUVEC cell proliferation and further the reduction of angiogen-esis in tumor tissues. This work could provide the foundation for the in-depth investigations on the phar-maceutical application of CIS targeting anti-tumor ther-apy.

Expression and purification of a fusion gene TSO45W-4B-TSOL18 of Taenia solium in Escherichia coli ArcticExpress(DE3) and preparation of rabbit antiserum / 中国地方病学杂志

Objective To construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18 of Taenia solium,to obtain purified recombinant protein TSO45W-4B-TSOL18 and to prepare rabbit antiserum against the recombinant fusion protein.Methods TSO45W-4B and TSOL18 encoding genes were connected with hydrophobic (Gly4Ser)3 linker.TSO45W-4B-TSOL18 fusion gene was synthesized and cloned into an expression vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18.The positive recombinants were transformed into Escherichia(E.) coli ArcticExpress (DE3),and the expression of recombinant protein was induced with isopropylβ-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).The expressed recombinant fusion protein was purified through affinity chromatography.The purified recombinant protein was mixed with complete Freunds adjuvant at a dosage of 0.5 mg.Rabbit was intramuscularly and subcutaneously immunized in the hind leg and the back,respectively.After 2 weeks the rabbit was boosted with purified recombinant protein which was mixed with incomplete Freunds adjuvant at the same dosage.Rabbit can be boosted every 10 days until an adequate response was achieved.At the 6th day after the last immunization,blood was collected from the rabbit heart.Serum was separated to purify and prepare the antiserum.ELISA was applied to determine the titer of the antiserum and Western blot assay was used to determine the specificity of the antiserum.Results The size of the synthesized fusion gene TSO45W-4B-TSOL18 was 789 bp.The results of restriction enzyme digestion showed that the recombinant plasmid pGEX-TSO45W-4B-TSOL18 was successfully constructed.DNA sequencing showed that the size of the fusion gene TSO45W-4B-TSOL18 was 789 bp,which was consistent with expected result.As demonstrated by SDS-PAGE,relative molecular mass of the expressed recombinant fusion protein was approximately 55 × 103,and its purity was 85％ after purification with affinity chromatography.Titer of the antiserum against the recombinant protein was 1 ∶ 512 000 in ELISA assay and the specific rabbit antiserum against the purified recombinant protein TSO45W-4B-TSOL18 could specifically bind to the recombinant fusion protein in Western blot assay.The relative molecular mass of the specific band was about 55 × 103,which was consistent with expected size.Conclusions The recombinant plasmid pGEX-TSO45W-4BTSOL18 of Taenia solium is successfully constructed.High quality recombinant fusion protein TSO45W-4B-TSOL18 and high titer rabbit antiserum are successfully prepared.

Establishment of serological detection methods of 3 types of human herpesvirus 8 specific-antigens and complex antigens / 中华传染病杂志

Objective To establish serological detection methods of human herpesvirus 8 (HHV-8).Methnds Three potent antigenic fusion proteins.K8.1,ORF65 and ORF73 C of HHV- 8 were synthesized using E.coli system.The sera were detected using lhese antigenic proteins.The positive sera were from 12 patients with Kaposi's sarcoma and 32 patients with acquired immunodeficiency syndrome-related Kaposi's sarcoma.The negative sera were from 20 patients with cutaneous tumors and children under 15 years old.Western blot and enzyme-linked immunosorbent assay (EI.ISA)were employed to determine the immunogenicity of each recombinant protein and the sensitivity and specificity of ELISA using the complex antigens.Results Three types highly purified HHV 8 specific recombinant pro teins with potent antigenicity were successfully synthesized.The sensitivity of ELISA using the above complex antigens was significantly higher than traditional immuno-flurescent assay (IFA)detecting the positive and negative sera,whieh were 81.8%,34.4%,respectively.And the specificity of ELISA was 97.9%.Conclusion K8.1,ORF65 and ORE73 C are good candidate antigens for establishing HHV-8 serological detection methods,which have better sensitivity and specificity.

Preparation and detection application of rat mAb against recombinant mouse Foxc2 / 中国免疫学杂志

Objective:In order to study the effect of forkhead box c2 (Foxc2) on the development of skeleton and aorta Methods:Rat monoclonal anti mouse Foxc2 antibody was prepared by using recombinant fusion protein GST Foxc2, and used it to detect the expression of Foxc2 protein in C2C12 myoblasts and embryo of mouse Results:The titer of rat anti mouse Foxc2 antibody was 1: 5 000 This monoclonal antibody can specifically recognized Foxc2 protein producing in mouse L cells and human bladder carcinoma HTB9 cells transfected with CX Foxc2 plasmid Endogenous Foxc2 protein was detected in the mouse myoblast C2C12 cells and increased significantly in C2C12 cells after treatment with bone morphogenetic protein 2(BMP 2) Endogenous Foxc2 protein level was lowered markedly by transfecting C2C12 cells with antisense Foxc2 sequence Foxc2 protein in 11 5 dpc embryo was localized in the developing selerotome and mesenchymal tissue around the aorta Conclusion:Rat anti mouse Foxc2 monoclonal antibody was successfully prepared by using recombinant fusion protein GST Foxc2 The BMP 2 induced Foxc2 protein may play an important role in regulating the osteoblastic differentiation of C2C12 myoblasts and in the formation of axial skeleton and aortic arch

Expression,purification and identification of hepatitis E virus pb166-GST fusion protein / 中华检验医学杂志

Objective To express,purify and identify recombinant hepatitis E virus(HEV) pb166-GST fusion protein using GST gene fusion system and investigate its potential role in researching Hepatitis E diagnostic antigen field.Methods The recombinant E.coli BL21 performed by our own laboratory was used to induce the HEV pb166-GST expression with IPTG.The products were purified by BD Biosience GST purifying system.The specific expression was identified by SDS-PAGE and Western blot.The experiment conditions and results were described and analysed.Results The resolved HEV pb166-GST fusion protein on SDS-PAGE showed a major band at position of 43 kD.The expressed proteins had a single expected band after purify and the protein was recognized by anti-GST antibody on PVDF membrane.Conclusion The recombinant HEV pb166-GST fusion protein is expressed in recombinant E.coli BL21 efficiently in this way,and might be used as a candidate for diagnostic antigen of HEV.

Expression and purification of the fusion hepatitis C virus hypervariable region 1 protein and it's preliminary application / 中华传染病杂志

Objective To clone and express hypervariable region 1 (HVR1) fragments of different genotypes of HCV strains from different cities of China.Then detect the reactivity of the expressed HVR1 fusion proteins with sera of chronic he-patitis C patients to analyzing it's signification in clinical utilization. Methods HVR1 genes of four HCV strains (genotype 1b and 2a) were amplified from pGEMT-E2 plasmids and cloned into pQE40 vectors, respectively to construct four recombinant prokaryotic expression plasmids which expressed HVR1 as fusion proteins with DHFR in Escherichia coli strain TG1. Then we used the purified DHFR-HVR1 proteins to detect the anti-HVR1 antibodies in 70 serum samples of chronic hepatitis C patients. Results Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320～ 800 ?g fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8%(51/70), 60%(42/70), 48.6%(34/70), and 58.6%(41/30) of the anti-HCV positive patients' sera, respectively by ELISA. 57% (4/7) of the non responders' sera taken before therapy reacted with all four HVR1 fusion proteins, while only 15.3% (2/13) of the sera from responders reacted with all of them. The A values of sera from IFN therapy responders were significantly higher than non responders (P