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目的原核表达人乳头瘤病毒(Human papilloma virus,HPV)58型截短型L1(Truncated L1,tL1)蛋白与结核分枝杆菌早期分泌型抗原靶蛋白-6(Early secreted antigenic target-6,ESAT-6)融合蛋白,并制备其抗血清。方法利用PCR技术分别从HPV基因组DNA和ESAT-6-18T质粒DNA中扩增tL1和ESAT-6基因,构建重组原核表达质粒ESAT-6-tL1-pET-21a,分别转化E.coli BL21(DE3)和Rosetta,IPTG诱导表达。表达的融合蛋白ESAT-6-tL1经镍离子亲和层析柱纯化后免疫昆明小鼠,ELISA检测血清抗体效价。结果 PCR扩增出750 bp的tL1基因片段和320 bp的ESAT-6基因片段,测序结果与GenBank登录的序列一致;重组表达质粒ESAT-6-tL1-pET-21a经双酶切鉴定,构建正确;表达的ESAT-6-tL1融合蛋白相对分子质量约40 000,在E.coli Rosetta中的表达形式为包涵体,纯化后纯度为85%,免疫小鼠血清抗体效价为1:4 000。结论成功表达了ESAT-6-tL1融合蛋白,并制备了其抗血清,为进一步研制检测HPV58的抗体奠定了基础。
Objective To detect the expression of Truncated L1 (tL1) protein of human papilloma virus (HPV) and the early secreted antigenic target-6 (ESAT- 6) Fusion protein and prepare its antiserum. Methods The tL1 and ESAT-6 genes were amplified by PCR from HPV genomic DNA and ESAT-6-18T plasmid respectively. The recombinant prokaryotic expression vector ESAT-6-tL1-pET-21a was constructed and transformed into E.coli BL21 ) And Rosetta, induced expression by IPTG. The expressed fusion protein ESAT-6-tL1 was purified by nickel ion affinity chromatography and then immunized Kunming mice. The serum antibody titers were detected by ELISA. Results The 750 bp tL1 gene fragment and 320 bp ESAT-6 gene fragment were amplified by PCR. The sequencing result was consistent with that of GenBank accession. The recombinant plasmid ESAT-6-tL1-pET-21a was identified by double enzyme digestion and was constructed correctly The relative molecular mass of the expressed ESAT-6-tL1 fusion protein was about 40,000. The expressed protein was expressed in E.coli Rosetta as an inclusion body with a purity of 85% after purification. The antibody titer of the immunized mouse serum was 1: 4,000. Conclusion The ESAT-6-tL1 fusion protein was successfully expressed and its antiserum was prepared, which laid the foundation for the further development of the antibody against HPV58.