Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells / 中国地方病学杂志

Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and cysteine protease inhibitor ( CPI ) gene from periodic Brugia malayi (Bm) and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 (+). pcDNA3.1 (+)/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE). Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass (Mr) of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell.

Cloning, sequencing of CPI gene from periodic Brugia malayi and prediction of B cell epitopes in its amino acid sequence / 中国地方病学杂志

Objective To clone and sequence the cysteine protease inhibitor gene of periodic Brugia malayi(BmCPI) and predict B-cell epitopes in amino acide sequence of BmCPI in order to provide basis for further study the expression of BmCPI and its function. Methods Total RNA was extracted from periodic Brugia malayi.A couple of specific primers were designed on the basis of known sequences of cysteine protease inhibitor gene from BmCPI. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli(E, coli) strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. Five parameters and methods were used to predict B-cell epitopes in amino acide sequence of BmCPI. Results For RT-PCR, a specific band of around 621 bp was amplified. The same band was obtained by double restriction of recombinant plasmids or PCR using recombinant plasmid as template. The result of DNA sequencing showed that BmCPI shares 99% nucleotide sequence identity with that of published sequence. It showed that B-cell epitopes were probably at or adjacent to 23 - 32, 50 - 79 and 117 - 126 in its amino acide sequence. Conclusions pGEM-BmCPI is successfully constructed and sequenced, anticipated objective is reached and conditions is provided for further study of BmCPI expression and its function.

Cloning,weukaryotic expremion of the gene encoding glyceraidehydes-3-phosphate dehydrogenase fromperiodic Brugia malayi / 中国地方病学杂志

Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase(GAPDH)from periodic Brugia molayi(Bm).Methods Total RNA was extraeted from periodic Brugic malayi.The BmGAPDH gene was amplified by RT-PCR.The PCR product was cloned and then subeloned into pcDNA3.1(+)vector.The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification,and were transformed into COS-7 cell subsequently.The expressed protein was identified by SDS-PAGE.Results BmGAPDH mRNA was highiy expressed in transfected COS-7 cell.The deduced amino acid sequence was identical with that of BmGAPDH.The recombinant pnotein wag about Nr 43 000.Conclusion The recombinant plasmid peDNA3.1(+)-BmGAPDH has been constructed and the protein has been expressed correctly.