Objective To explore enhanced specific immune response in BALB/c mice immunized with codon-modified human papillomavirus type 6b(HPV6b)L1 DNA.Methods A wild-type(wt)HPV6b LI gene was extracted from tissue samples of patients with condyloma acuminata and inserted into eukaryotic expression vector pcDNA3.1(+)to construct a recombinant plasmid pcDNA3.1(+)/HPV6bL1(wt).In addition,the HPV6b L1 gene(wt)was optimized according to codon bias in eukaryotic genes.Then the HPV6b L1 gene(mod.)sequence was obtained by overlap PCR and inserted into pcDNA3.1(+)to construct peDNA3.1(+)/HPV6bL1(mod.).Six to eight week old female BALB/c mice were divided into 4 groups and intramuscularly injected with pcDNA3.1(+)/HPV6bL1(mod.),pcDNA3.1(+)/HPV6bL1(wt),pcDNA3.1 (+) and PBS,respectively,every 2 weeks for 3 times.Serum samples were collected at 0,2,4,6 and 8 weeks and specific IgG antibodies were detected with indirect ELISA.The activity of specific CTL was measured at 8 weeks to detect cellular immune response at effector-to-target (E/T) ratios of 5:1,10:1 and 20:1,respectively.Results The codon-modified HPV6b L1 gene was inserted into pcDNA3.1 (+) success fully.The specific IgG titers increased with time and the number of immunization.The titer reached a peak at 8 weeks in pcDNA3.1 (+)/HPV6bL1 (mod.) group and was significantly higher than those in pcDNA3.1 (+)/HPV6bL1 (wt),peDNA3.1 (+) and PBS groups (t=18.138,t=29.140,t=30.840,P＜0.01),respectively. There was also statistically significant difference between pcDNA3.1 (+)/HPV6bL1 (wt) group and the groups of pcDNA3.1 (+) and PBS (t=20.072,t=22.457,P＜0.01).The CTL activities in pcDNA3.1 (+)/HPV6bL1 (mod.) group were significantly higher than those in pcDNA3.1(+)/HPV6bL1 (wt) group (t=11.892,t=15.329,t=2.911,P＜0.05),peDNA3.1 (+) group (t=12.936,t=16.613,t=13.901,P＜0.01) and PBS group (t=14.768,t=18.935,t=10.925,P＜0.01) at the E/T ratios of 5:1,10:land 20:1,respectively.Conclusion Codon-modified HPV6b LI DNA can induce stronger specific humoral and cellular immune response in BALB/e mice.%目的 探讨真核细胞偏好密码子优化对人乳头瘤病毒(human papillomavirus,HPV)6b型L1 DNA诱导BALB/c小鼠特异性体液和细胞免疫应答的增强效应.方法 利用PCR从尖锐湿疣组织中扩增HPV6b L1基因,将其克隆至真核表达质粒pcDNA3.1(+),构建野生型pcDNA3.1(+)/HPV6bL1(wt)并进行鉴定;同时,对HPV6b L1基因进行真核细胞偏好密码子优化,进一步通过重叠PCR的方法获得全长基因,构建密码子优化的重组质粒pcDNA3.1(+)/HPV6bL1(mod.)并进行鉴定.将6～8周龄雌性BALB/c小鼠分成4组,分别肌肉注射3次pcDNA3.1(+)/HPV6bL1(mod.)、pcDNA3.1(+)/HPV6bL1(wt)、pcDNA3.1(+)载体和PBS,间隔2周,每次剂量为150μg/鼠.分别于免疫前和免疫后每隔2周,收集血清,用间接ELISA检测血清igG抗体;并于第8周取小鼠脾细胞,采用乳酸脱氢酶释放法,按效应细胞与靶细胞之比(E:T)为5:1、10:1、20:1进行免疫小鼠特异性CTL杀伤活性检测.结果 成功构建了密码子优化的HPV6b L1重组质粒.小鼠免疫后血清特异性IgG抗体水平随着免疫时间和次数的增加而升高.pcDNA3.1(+)/HPV6bL1(mod.)组血清IgG抗体在第8周达到高峰,与pcDNA3.1(+)/HPV6bL1(wt)、载体和PBS对照组相比差异均有统计学意义(t=18.138、t=29.140、t=30.840,P值均＜0.01);而pcDNA3.1(+)/HPV6bL1(wt)组与载体和PBS组比较差异也有统计学意义(t=20.072、t=22.457,P值均＜0.01).在特异性CTL杀伤实验中,当E:T分别为5:1、10:1、20:1时,pcDNA3.1(+)/HPV6bL1(mod.)组的脾细胞特异性CTL杀伤率明显高于pcDNA3.1(+)/HPV6bLl(wt)组(t=11.892、t=15.329、t=2.911,P值均＜0.05)、载体组(t=12.936、t=16.613、t=13.90l,P值均＜0.01)和PBS对照组(t=14.768、t=18.935、t=10.925,P值均＜0.01).结论 密码子优化的HPV6b L1 DNA可诱导BALB/c小鼠产生增强的特异性体液和细胞免疫应答.
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