Preparation,identification and comparison of murine and rabbit polyclonal antibodies against matrix protein of rabies virus strain CVS-11 / 中国生物制品学杂志

@#Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV) matrix(M) protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA) and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1:100 and 1:256 000respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.

Cloning, bioinformatics, and expression analysis of MpF2H gene in Microcos paniculata / 中草药

Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.

Cloning and expression pattern analysis of chalcone synthase gene in Microcos paniculata / 中草药

Objective To clone the full-length cDNA of MpCHS and analyze its expression pattern in different parts of Microcos paniculata and different growth periods of M. paniculata leaves. Methods Based on the transcriptome data of M. paniculata, we designed specific primers for MpCHS gene. The full-length cDNA of MpCHS was amplified by PCR and the positive clones were then sequenced, analyzed, and constructed prokaryotic expression vector. The bioinformatics analysis of MpCHS was also performed. Meanwhile, the mRNA expression of MpCHS was detected using real-time quantitative PCR. Results The relative molecular mass was 42 700, and its theoretical isoelectric point was 6.11, with three conserved functional active sites (165 C, 304 H, and 337 N) of the CHS family proteins and the tag sequence of RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Phylogenetic tree analysis showed that MpCHS had close relationship with woody plants such as cocoa and upland cotton. We successfully cloned the full-length cDNA of MpCHS (GenBank: KY472608). It had an ORF of 1 176 bp which encoded a protein of 391 amino acid residues. RT-qPCR results showed that MpCHS was expressed in all parts of M. paniculata and its expression in leaves was gradually decreased along with its development. Conclusion MpCHS is cloned from M. paniculate for the first time, and the gene expression pattern of MpCHS in different parts of M. paniculata and different growth periods of M. paniculata leaves was analyzed. This study facilitates the further purification and functional validation of MpCHS protein and provides reference for further analysis of flavonoids biosynthesis pathway in M. paniculata.

Expression and characterization of recombinant flagellin FlgE of Pseudomonas aeruginosa / 中华微生物学和免疫学杂志

Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.

Preparation of the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 and its application / 中国人兽共患病学报

To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.

Anti-inflammatory action of recombinant mouse HMGB1-A box in RAW264.7 cells / 中国免疫学杂志

Objective: To study the influence of HMGB1-Abox on the expression of HMGB-1 in RAW264.7 cells stimulated by lipopolysaccharide(LPS)and its inhibition on the secretion of inflammatory factor.Methods: Use cloning vector to make up prokaryotic plasmid PET28a-HMGB1-Abox, which included the gene fragment of the A box of HMGB1, and recombinant vector was further transformed into Escherichia coil strain BL21(DE3),the recombinant plasmid was induced by isopropylthiogalactoside(IPTG)to express target protein.The protein was purified by Ni-column.We tested the effect of the different concentration of rHMGB1-Abox on the viabihty of RAW264.7 cells stimulated by LPS using CCK-8.Experimental group(EG)was treated with rHMGB41-Abox and control group(CG)was deh with PBS.TNF-α and IL-1β levels were detected by the enzyme linked immunosorbent assay(ELISA).Western blot and immunofluorescence staining method were used to compare the expression of HMGB1 in RAW264.7 cells with the experiment group and control group.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the variation tendency of HMGB1-mRNA.All procedures were manipulated at 2 \ 6 \ 12 \ 24 \ 48 h after treatment.Results: The recombinant prokaryotic expression vector PET28a-HMGB1-Abox was successfully constructed and mouse HMGB1-Abox protein about 14 kD was successfully expressed.There was a positive correlation between the viability of RAW264.7 cells and the concentration of protein and acting time.Compared to CG, TNF-α and IL-1β levels in EG declined significantly.In EG, Western blot and Immunofluorescence staining showed that the expression of HMGB1 protein was decreased and the expression of HMGB1-mRNA was reduced markedly and delayed.Conclusion: The rHMGB1-Abox could reduce expression and secretion of HMGB1 in RAW264.7 cells stimulated by LPS significantly,thereby to prevented the promotion of HMGB1 on pro-inflammatory mediator and inhibit the expression and secretion of inflammatory factors,which has some anti-inflammatory action.

Construction of expression vector for NT4-ADNF-9 fusion gene / 西安交通大学学报·英文版

Objective: To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods: By means of asymmetrical primer/ template, double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results: Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3′terminal of the signal and leader peptides of NT4, and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion: Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.

Construction of expression vector for NT4-ADNF-9 fusion gene / 药物分析学报

Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neuraseusory deafness. Methods By means of asymmetrical prince/ template, double stranded eDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 eDNA was ligated to the signal and leader peptides of nenrotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was suheluned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restrtction enzymes digestion showed that we recombined ADNF-9 eDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcluned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 cbicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivtty is satisfactory.

Construction of a prokaryotic expression vector for Apoptin and expression in E.coli / 西安交通大学学报(医学版)

Objective To construct an Apoptin prokaryotic vector,aiming to produce antigenic fusion protein Apoptin. Methods The Apoptin gene was amplified from the template of plasmid pSSCHG/NT4-Apoptin-HA2-TAT by PCR.The Apoptin was sub-cloned into the multiple clone sites of plasmid pET-28a(+) to get the prokaryotic vector of pET-28a(+)-Apoptin,which was transformed into E.coli BL21(DE3).Expression of E.coli BL21(DE3) was induced by IPTG.The specific protein expression was detected by SDS-PAGE. Results The fusion protein was expressed with high efficiency in E.coli BL21(DE3) transformed by pET-28a(+)-Apoptin after induction with IPTG.The specific fusion protein had an apparent related molecular weight of about 17 000 ku as indicated by SDA-PAGE analysis. Conclusion The Apoptin prokaryotic expression vector with pET-28a(+)-Apoptin can effectively express Apoptin fusion protein,laying a foundation for further study of Apoptin and preparation of antibodies against Apoptin.

The construction and identification of prokaryotic expression vector pQE30-EWS-FLI1 / 重庆医科大学学报

Objective:To construct Ewing's sarcoma EWS-FLI1 gene prokaryotic expression vector.Methods:The target gene of EWS-FLI1 was obtained by RT-PCR method after the total RNA was extracted from Ewing's sarcoma A673 cells.The site sequences of restrictive endonuclease SacⅠand Hind Ⅲ were introduced into the upstream and downstream of target gene respectively.The target gene fragment were cloned into pMD18-T and transformed into E.Coli JM109.Screened positive clones were confirmed by PCR,restrictive endonuclease digestion and DNA sequencing.The EWS-FLI1 gene was sequentially subcloned into prokaryotic expression vector pQE30,and the recombinant plasmid pQE30-EWS-FLI1 was confirmed by restrictive endonuclease digestion and DNA sequencing.The proteins,expressed in E.coli JM109 transformed with EWS-FLI1recombinant plasmid under IPTG induction,were characterized by SDS-PAGE and Western-blot.Results:PCR result indicated that an amplified DNA fragment was in size of 1.5 kb.Restrictive endonuclease digestion analysis indicated that the target gene was in size of 1.5 kb.DNA sequencing analysis demonstrated that sequence of target gene accorded with anticipated one.The EWS-FLI1 with a molecular weight of 54 kD was highly expressed in pQE30-EWS-FLI1.Western blot proved that the expressed product had the antigenicity of EWS-FLI1.Conclusion:The recombinant prokaryotic expression vector pQE30EWS-FLI1 is constructed successfully,which will contribute to the further research of EWS-FLI1.

Cloning and onstruction of recombinant vector of bivalent vaccine candidate of human Helicobacter pylori / 重庆医科大学学报

Objective:To construct a recombinant vector containing gene encoding heat shock protein A (HspA) and VacA with Mr 13 000 and 26 000 simultaneously from human Helicobacter pylori(Hp)for study on expression in E.coli BL21,and analysis of their antigenicity.Methods:The target genes encoding HspA and VacaA were amplified from Hp chromosome by PCR,digested by restricted endonuclease enzyme respectively,and inserted into the corresponding enzyme digested prokaryotic expression vector pET32a(+).The recombinant vectors pET32a(+)/HspA and pET32a(+)/VacA were used to select and transform for sequence analysis.After recombinant vectors digested by restricted enzyme of Xhol,BamH Ⅰ simultaneously,the pET32a(+)/HspA and VacA were taken out of agarose electrophoresis,and connected by T4 ligase again.The recombinant vector pET32a(+)/HspA-VacA was used to select and identified by PCR and restricted endonuclease enzyme.Results:Enzyme digestion analysis and sequencing showed that the target genes were found in 1080 base pairs,and had been inserted into recombinant vector,but as compared with gene reported by GenBank,3.40% of the gene mutation and 1.11% of amino acid residues change in Hp happened respectively.Conclusion:The genes coding HspA and VacA with Mr 13 000 and 26 000 respectively are cloned successfully.The results obtained lay the foundation for research on development of Hp protein and DNA vaccine applying to prevention of Hp infection.

High efficient expression of GST-hureb1 and generation of anti hureb1 antibody / 中国免疫学杂志

Objective:To construct high level prokaryotic cell expression vector carrying human upstream regulatory element binding protein1 (hureb1) gene for producing GST hureb1 fusion protein and generating anti hureb1 antibody Methods: After the Xho I/Not I fragment from hureb1 open reading frame was recombined into the prokaryotic expression vector pGEX 4T 2, the vector was transformed into E coli BL21(DE3) strain and induced with IPTG to express the fusion protein, GST hureb1 The crudely isolated fusion protein was applied to immunize rabbit and mouse for generating anti hureb1 polyclonal antibody and monoclonal antibody respectively Results:The prokaryotic expression vector expressing GST hureb1 fusion protein was constructed and induced to produce high level GST hureb1 that was detected up to 33 45% of the total bacterial protein expressed The GST hureb1 fusion protein immunizing animals generated high titer and specific anti hureb1 antibodies Conclusion:Recombinant hureb1 protein and anti hureb1 antibodies can be used to analyze the biological functions of hureb1