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目的表达人乳头瘤病毒16型(HPV16)E2蛋白,并制备小鼠抗HPV16 E2血清。方法采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物。经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白。免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4~+T细胞、CD8~+T细胞、CD4/CD8比值和抗血清滴度变化。结果酶切和测序结果表明pET21b-HPV16 E2构建成功。表达蛋白相对分子质量(M_r)为42 000,Western blot法证明具有较高特异性。小鼠抗血清效价升高,CD4~+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高。结论成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清。
Objective To express human papillomavirus type 16 (HPV16) E2 protein and prepare mouse anti - HPV16 E2 serum. Methods The HPV16E2 gene was amplified by PCR and inserted into pET21b vector. The recombinant plasmid pET21b-HPV16E2 was identified and transformed into E. coli BL21 (DE3) to express and identify the expression product. The purified HPV16 E2 protein was prepared by purification, denaturation and refolding methods. BALB / c mice were immunized to prepare antisera. The changes of IFN-γ, CD4 ~ + T cells, CD8 ~ + T cells, CD4 / CD8 ratio and antiserum titers in mice were detected. Results The results of enzyme digestion and sequencing showed that pET21b-HPV16 E2 was successfully constructed. The relative molecular mass (M_r) of expressed protein was 42 000, which was proved to be highly specific by Western blot. The titer of mouse antiserum increased, the number of CD4 ~ + T cells and the ratio of CD4 / CD8 increased, and the level of IFN-γ in mouse did not increase. Conclusion The high titer anti-serum of HPV16 E2 protein and mouse anti-HPV16 E2 were successfully prepared.