Zhao Li, Gao Meng, Gao Jian, Ren Jiao, Zhang Hui, Tian Hou-wen, Tan Wen-jie, Ruan Li
National Institute for Viral Disease Control and Prevention, Beijing, China.
Zhonghua Zhong Liu Za Zhi. 2012 Nov;34(11):810-5. doi: 10.3760/cma.j.issn.0253-3766.2012.11.003.
To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.
The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.
The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.
The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.
通过原核表达系统研究HPV16L2N120E7E6融合蛋白的高表达，并评估其在接种小鼠中的免疫原性和抗肿瘤疗效。
通过重叠延伸PCR构建HPV16L2N120E7E6融合基因，对其密码子进行优化以增加蛋白表达，然后将其插入原核表达载体pET9a。接着，在携带质粒pETL2N120E7E6的大肠杆菌BL21(DE3)菌株中用IPTG诱导表达融合蛋白，并通过SDS-PAGE和Western-blot进一步检测。最后，分别用ELISA和ELISPOT检测接种纯化的HPV16L2N120E7E6融合蛋白的小鼠的体液免疫和细胞免疫反应，并使用TC-1肿瘤攻击模型评估小鼠的抗肿瘤疗效。
密码子优化的HPV16L2N120E7E6融合基因在携带质粒pETL2N120E7E6的大肠杆菌BL21(DE3)菌株中高表达，融合蛋白量占细菌总蛋白的近48.6%。纯化的融合蛋白可在接种小鼠中诱导针对L2、E7和E6的高滴度特异性抗体。当与佐剂CpG联合使用时，融合蛋白能够分别在接种小鼠中引发针对肽HPV16E7(49-57)和HPV16E6肽池的强烈和中等程度的细胞免疫反应。此外，肿瘤治疗实验表明，HPV16L2N120E7E6 + CpG可使80.0%（8/10）接种小鼠预防肿瘤形成。
本研究数据表明，HPV16L2N120E7E6融合蛋白可能是治疗慢性HPV16感染和宫颈癌术后辅助治疗的有前景的候选疫苗。
