带有不同亚型HPVL_1基因片段的哺乳动物细胞克隆的建立

Establishment of mammalian cell cloning with different subtype HPV L_1 gene fragment

目的构建携带HPV亚型L1基因片段的标准细胞株。方法根据HPV通用引物GP5+/GP6+,用重叠延伸PCR方法合成HPV亚型L1基因中约150bp片段;插入真核细胞表达载体EGFPN1,用脂质体转染SPC细胞,筛选以获得带有不同HPVL1基因片段细胞克隆,并用定量PCR方法确认。结果重叠延伸PCR后得到约150bp的基因片段,测序结果经BLAST比对,分别与相应HPV亚型公开报告序列一致;将所获片段与EGFPN1载体连接后酶切及测序鉴定正确;经筛选获得了携带HPV6、11、16、18、31、35亚型L1基因片段的细胞株;并用定量PCR方法进行了确认。结论成功构建了带有不同HPV亚型L1基因片段的30个亚克隆和6个细胞株,为进一步的HPV分型检测研究提供了保证。

Objective To prepare the standard cell line involved clinical frequent HPV subtype L1 gene fragment. Methods According to HPV consensus primer GP5^＋/GP6^＋ , 150 bp gene fragment of each HPV subtype L1 gene was synthesized with the overlap extension PCR, recombined with PMD18-T vector and identified by enzymase digestion and sequencing. The recombinant was restructed with EGFP-N1 vector, transfected into SPC cell line, screened by G418 and identified by real-time PCR. Results A gene fragment about 150 bp was obtained by OE-PCR, and its DNA sequence was identical to public sequence of each subtype. Each recombinant plasmid was conjuncted with EGFP-N1 vector and confirmed by enzymase digestion and sequencing. Screened by G418, cell strains involving HPV6, 11, 16, 18, 31,35 L1 gene fragment were obtained and identified by real-time PCR. Conclusion The successful cloning of 30 clinical frequent HPV subtype LI gene fragments and construction of 6 cell strains have laid a foundation for the further studies on detection of HPV.