【摘要】 目的构建人乳头瘤病毒11型(HPV11)L1原核表达系统pBAD(B)鄄HPV11L1,表达HPVL1蛋白,为进一步制备基因工程疫苗奠定基础。方法PCR法从尖锐湿疣组织标本中扩增人乳头瘤病毒11型L1基因片段,克隆至pBluescript质粒,并测序。将其从克隆重组质粒中切下连入表达载体,建立pBAD(B)鄄HPV11L1原核表达重组质粒,在大肠杆菌宿主菌Top10中,经阿拉伯糖诱导,表达融合蛋白L1,经SDS鄄PAGE电泳和Westernblot进行鉴定。结果3株HPV11L1经测序发现变异一致,每株均有2个核苷酸变异,但未影响氨基酸变化。经SDS鄄PAGE鉴定L1蛋白相对分子质量为59×103,与预期值相同,Westernblot证明表达蛋白能与单克隆抗体反应,成功构建了pBAD(B)鄄HPV11L1原核表达系统。结论所构建的pBAD(B)鄄HPV11L1原核表达系统能够高效表达HPV11L1蛋白。更多还原

【Abstract】 Objective To develop the genetic engineering vaccine of human papillomavirus (HPV) construction and expression of HPV type11 was made and expressed in the E. coli prokaryotic expression system. Methods The full length of HPV 11 L1 coding region was amplified from condyloma acuminate tissues by using PCR. The amplified products were cloned into plasmid pBluescript and sequenced. Subsequently, the recombinant expression vector pBAD(B)-HPV11L1 was constructed by inserting the HPV11 L1gene into pBAD(B). The L1 protein was expressed in host strain top 10 by L-Arabinose inducing and identified by using SDS-PAGE and Western blot. Results Three clones of HPV11 L1 were established. Sequence analysis showed that all the three L1 genes have two nucleotide variants at the same positions, C258T and C837T, comparing with the HPV type 11 prototype. The mutation did not affect the corresponding amino acid sequences. SDS-PAGE and Western blot showed the expressed protein of 59×103 specifically responds to the monoclonal antibody against HPV11 L1. Conclusions We have successfully constructed expressing vector pBAD(B)-HPV11 L1 and expressed the L1 protein in the prokaryotic system with high quantity.更多还原