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目的将含周期型马来丝虫3-磷酸甘油醛脱氢酶(BmGAPDH)和半胱氨酸蛋白酶抑制剂(BmCPI)复合基因的重组质粒pcDNA3.1(+)-BmCPI/BmGAPDH转染人宫颈癌细胞(Hela)细胞获得复合重组质粒稳定转染细胞株,并纯化所表达的重组蛋白,为研制新型的抗周期型马来丝虫重组蛋白疫苗提供理论及实验依据。方法将成功构建的pcDNA3.1(+)-BmCPI/BmGAPDH和空载体pcDNA3.1(+)分别转染Hela细胞,以G418筛选转染细胞,进而通过RT-PCR和SDS-PAGE鉴定G418筛选后的单克隆抗性细胞株。对阳性克隆进行无血清悬浮培养,收集细胞及其培养液。亲和层析纯化所表达的重组蛋白并通过Western-blotting对纯化的重组蛋白进行生物学鉴定。结果重组真核表达质粒pcDNA3.1(+)-BmCPI/BmGAPDH经转染Hela细胞,G418持续筛选14 d获得稳定表达。表达产物经Western-blotting法鉴定能够与相应的免疫鼠血清反应,相对分子质量Mr约为54×103。结论成功建立了周期型马来丝虫复合重组质粒pcDNA3.1(+)-BmCPI/BmGAPDH稳定转染细胞株并获得了相应的重组蛋白。
OBJECTIVE: To construct a recombinant plasmid pcDNA3.1 (+) - BmCPI / BmGAPDH containing a combination of BmGAPDH and BmCPI, The recombinant plasmid was successfully transfected into Hela cells, and the expressed recombinant protein was purified. The results provided theoretical and experimental evidences for the development of a new type of anti-cycle Malayan worm recombinant protein vaccine. Methods HeLa cells were transfected with pcDNA3.1 (+) - BmCPI / BmGAPDH and empty vector pcDNA3.1 (+) respectively. The transfected cells were selected by G418, and then identified by RT-PCR and SDS-PAGE. Of monoclonal anti-cell lines. Positive clones were cultured in serum-free suspension and the cells and culture medium were collected. The expressed recombinant protein was purified by affinity chromatography and the purified recombinant protein was identified by Western-blotting. Results The recombinant eukaryotic expression vector pcDNA3.1 (+) - BmCPI / BmGAPDH was transfected into Hela cells. G418 was screened for stable expression for 14 days. The expressed product was identified by Western-blotting with the corresponding immune mouse serum reaction, the relative molecular mass Mr about 54 × 103. Conclusion The recombinant plasmid pcDNA3.1 (+) - BmCPI / BmGAPDH was successfully established and the corresponding recombinant protein was successfully constructed.