HPV-16-11 L1双价重组杆状病毒的构建及VLP形成

Coexpression of HPV-16 and -11 L1 genes using the bivalent recombinant baculovirus and VLPs assembled by L1 proteins

目的将本地区流行株HPV-16 L1基因与HPV-11 L1基因双表达于杆状病毒昆虫细胞表达系统,组装成类病毒颗粒(virus like particles,VLP)。为研制同时预防宫颈癌和尖锐湿疣等多种疾病的HPV-16-11型基因工程疫苗奠定基础。方法对本地流行株HPV-16 L1基因进行亚克隆,构建pBluescript-HPV-16L1克隆质粒,并对其测序。再将质粒中HPV-16 L1基因切下,连接到pFastBacDual的强启动子phol后;从克隆质粒pSP73-HPV-11 L1切下HPV-11 L1,连接到pFastBacDual的弱启动子p10后,利用Bac to Bac系统在昆虫细胞Sf9中表达外源蛋白。SDS-PAGE和Western blot鉴定HPV-16 L1和HPV-11 L1蛋白的表达量及特异性,电镜观察昆虫细胞内VLP的存在。结果酶切和PCR法证实pFastBacDual-HPV-16 L1-HPV-11 L1转移质粒连接正确,转染昆虫细胞Sf9后,检测到HPV-16和HPV-11 L1特异蛋白,两种蛋白的相对分子质量(Mr)相近,约56×103;电镜观察到了VLP。结论本研究利用Bac to Bac系统构建Bacmid／HPV-16-HPV-11 L1,转染到Sf9昆虫细胞后重组外源蛋白得到高效表达,并经电镜证实昆虫细胞内存在较大量VLP,用此VLP免疫BALB／c小鼠后产生抗L1蛋白的抗体。共表达HPV L1蛋白可能为预防多型别HPV感染提供新途径。

Objective To make HPV-16-11 bivalent engineering vaccine coexpression of HPV-16 and -11 L1 using the bivalent recombinant haculovirus and Vires-like particles （VLPs） assembled by the L1 proteins. Methods The locally prevalent HPV-16 L1 gene was sub-cloned and pBluescript-HPV-16L1 was constructed and sequenced. The HPV-16 L1 gene was inserted into the downstream of the strong promoter pho1. The locally prevalent HPV-11 L1 gene from pSP73-HFV-11L1 plasmid was inserted into the downstream of the weak promoter p10 of baculovirus shuttle vector pFastBacDual. The HPV-16 and HPV-11 L1 proteins were coexpressed in Bac-to-Bac baculovirus insect cell Sf9 expression system and were identified by Western blot. VLP in insect cells were identified by transmission electron microscope （TEM）. Results Baculovirus shuttle vector pFastBacDual-HPV-16L1-HPV-11L1 was correctly constructed. Then, the recombinant baeulovirus Bac/HPV-16L1-11L1 was generated by action of transpon in E.coli strain, DH10Bac. The bacmid/HPV-16-HPV-11L1 was generated. HPV-16 L1 and -11 L1 proteins from the insect cells Sf9 infected with the recombinant baculovirus were identified and a molecules relative mass（Mr） 56×10^3 hand composing both HPV-16 and -11 proteins was shown in SDS-PAGE and Western blot. TEM observation showed abundant VLPs in the Sf9 cells. Conclusion We have successfully construoted the recombinant haculovirus Bac/HPV-16L1-11L1 using locally prevalent HPV-16 and-11. TEM results indicated that the proteins assembled into VLPs. After immunization using VLPs, the anti-L1 proteins antibodies were detected in serum of immunized BALB/c mice. The coexpression of the proteins of different HPV types may provide new tools for prevention of HPVs infection.