CAJ | 학술논문

目的在原核表达系统高效表达人乳头瘤病毒(HPV) 16型L2N120E7E6融合蛋白,获得具有高表达水平和强免疫原性的HPV治疗性融合蛋白疫苗.方法利用重叠PCR方法构建经密码子优化的HPV16L2N120E7E6融合基因,然后将其插入pET9a原核表达载体中,采用Western blot 法对表达蛋白进行鉴定.对HPV16L2N120E7E6融合蛋白免疫的小鼠进行特异性抗体和细胞免疫反应的检测,评价其对肿瘤生长的抑制作用.结果密码子优化的HPV16L2N120E7E6基因能在原核表达载体pET9a中高效表达,表达水平约占全菌蛋白的48.6％.HPV16L2N120E7E6融合蛋白免疫的小鼠可检测到针对L2、E7和E6的高滴度抗体,抗体滴度分别为1:40317、1:16000和1:800.CPG1826佐剂和HPV16L2N120E7E6融合蛋白免疫小鼠后,可产生较强的针对HPV16E749-57肽特异性T细胞免疫反应和较弱的E6特异性T细胞免疫反应,其分泌γ-干扰素的效应T细胞斑点数分别为(464.00±35.52)个和(47.00±11.67)个.HPV16L2N120E7E6融合蛋白对小鼠移植瘤生长有明显的抑制作用,有80.0％(8/10)的小鼠不成瘤.结论 HPV16L2N120E7E6融合蛋白可作为治疗HPV16慢性感染和宫颈癌手术后辅助治疗的候选疫苗.
목적 재원핵표체계통고효표체인유두류병독(HPV) 16형L2N120E7E6융합단백,획득구유고표체수평화강면역원성적HPV치료성융합단백역묘.방법 이용중첩PCR방법구건경밀마자우화적HPV16L2N120E7E6융합기인,연후장기삽입pET9a원핵표체재체중,채용Western blot 법대표체단백진행감정.대HPV16L2N120E7E6융합단백면역적소서진행특이성항체화세포면역반응적검측,평개기대종류생장적억제작용.결과 밀마자우화적HPV16L2N120E7E6기인능재원핵표체재체pET9a중고효표체,표체수평약점전균단백적48.6％.HPV16L2N120E7E6융합단백면역적소서가검측도침대L2、E7화E6적고적도항체,항체적도분별위1:40317、1:16000화1:800.CPG1826좌제화HPV16L2N120E7E6융합단백면역소서후,가산생교강적침대HPV16E749-57태특이성T세포면역반응화교약적E6특이성T세포면역반응,기분비γ-간우소적효응T세포반점수분별위(464.00±35.52)개화(47.00±11.67)개.HPV16L2N120E7E6융합단백대소서이식류생장유명현적억제작용,유80.0％(8/10)적소서불성류.결론 HPV16L2N120E7E6융합단백가작위치료HPV16만성감염화궁경암수술후보조치료적후선역묘.
Objective To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system,and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.Methods 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.Results 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 HPV16E749-57 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.Conclusions 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.