摘要
目的构建重组原核表达质粒获得人乳头瘤病毒18型(HPV18)主要衣壳蛋白,为进一步研制HPV18基因工程疫苗打下基础。方法以重组质粒pBR322-HPV18为模板,利用PCR方法扩增HPV18L1 DNA片段,将HPV18L1 DNA与pUC 19质粒重组构建pUC19-HPV18L1重组质粒;用酶切电泳验证重组结果的正确性;并通过测序检查质粒重组后序列有无变化;再利用pQE32质粒做表达载体构建重组质粒pQE32-HPV18L1,并用酶切电泳验证;利用SDS-PAGE检测HPV18L1目的蛋白;利用Western杂交鉴定HPV18L1目的蛋白的特异性。结果PCR扩增DNA片段约为1.7kb,与预期结果相同;克隆重组质粒pUC19-HPV18L1酶切后显示的酶切图谱与预期相同,而且测序验证插入片段全序列无改变;表达重组质粒pQE32-HPV18L1酶切图谱亦与预期相同;SDS-PAGE显示,约63×103处可见目的蛋白带,与预期结果一致;Western杂交鉴定,在Mr约63×103处可见目的条带,与预期结果一致。结论成功构建原核表达重组质粒pQE32-HPV18L1并能表达HPV18主要衣核蛋白。
OBJECTIVE To develop recombinant HPV18 vaccine by prokaryotic expression strategy. METHODS The L1 gene of HPV18 was amplified by PCR from pBR322-HPV18 and cloned into pUC19. The sequence of cloned HPV18 L1 was confirmed by restriction analysis and DNA sequencing. A HPV18L1 prokaryotic expression plasmid,pQE32-HPV18L1,was then constructed by subclone, pQE32-HPV18L1 transformed E. coli M15 （with pREP4） was induced by IPTG. The expression of L1 protein was analyzed by SDS-PAGE and Western blot.RESULTS The amplified DNA fragment was in size of 1.7kb as expected. Restriction analysis showed that the amplified gene was inserted in pUC19 correctly, Sequence showed there was no mutation in both ends of the cloned L1. Restriction analysis showed that the recombinant pQE32-HPV18L1 was right. A Mr 63 × 10^3 protein could be seen in M15 induced by IPTG with SDS-PAGE and was positive while reacting with HPV18 L1 antibody by Western blot. CONCLUSIONS HPV18L1 is successfully cloned and expressed in this study.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2005年第8期845-848,共4页
Chinese Journal of Nosocomiology
关键词
人乳头瘤病毒18型L1基因
基因重组
蛋白表达
鉴定
Human papillomavirus type 18L1 gene
Recombinant gene
Protein expression
Identification
