HIV-1B`/C、A／E重组亚型pol基因DNA疫苗的构建及其免疫原性研究  

Immunogenicities and comparison of DNA vaccines encoding pol genes derived from BVC and A/E recombinant HIV-1 strains

作　　者：万延民[1] 任艳琴[1] 王婧[1] 任晓楠[1] 胡志东[1] 仇超[2] 徐建青[2] 

机构地区：[1]上海市公共卫生临床中心科学研究部,201508 [2]复旦大学生物医学研究院

出　　处：《中华预防医学杂志》2012年第6期551-555,共5页Chinese Journal of Preventive Medicine

基　　金：基金项目：国家自然科学基金（81072496H1014）;上海市自然科学基金（11ZR1430600）

摘　　要：目的构建表达中国BYC和A／E重组亚型HIV-1pol基因的DNA疫苗，并比较其免疫原性。方法按哺乳动物密码子使用频率对HIV-1CN54（BYC重组亚型）与AE2f（A／E重组亚型）的pol基因进行序列优化，构建DNA疫苗，通过Westernblotting验证其体外表达效率。通过肌肉注射途径免疫小鼠，然后无菌分离小鼠脾淋巴细胞并分别在ConsensusBPol和HIV-1AE2f Pol肽库刺激下，采用酶联免疫斑点技术（ELISPOT）检测抗原特异性IFN-γ的分泌情况，观察比较两个DNA疫苗免疫原性特征。结果DNA测序结果表明两个pol基因重组质粒构建正确，且两个DNA疫苗均能在体外有效表达。ConsensusBPol肽库刺激后，pSVAE—Pol活化的总T细胞免疫反应为（636±178）个斑点形成细胞数／10。个脾淋巴细胞，pSVCN—Pol活化的总T细胞免疫反应为（468±265）个斑点形成细胞数／10^6个脾淋巴细胞（P=0．412）；HIV-1 AE2fPol肽库刺激后，pSVAE—Pol活化的总T细胞免疫反应为（1378±611）个斑点形成细胞数／10^6个脾淋巴细胞，pSVCN—Pol活化的总T细胞免疫反应为（713±61）个斑点形成细胞数／10^6个脾淋巴细胞（P=0．134）。将总T细胞反应按肽池分解后发现，pSVAE—Pol活化的特异性T细胞反应主要集中于Pol 1肽池；而pSVCN—Pol除了能够活化针对Pol1的T细胞反应之外，也可以较好地活化针对ConsensusBPol2肽池的T细胞反应。结论两个亚型PolDNA疫苗中以AE-Pol的免疫原性更强，但其所活化的T细胞表位识别谱不及CN—Pol广泛。HIV-1疫苗可能需将两者结合在一起。Objective To construct and compare the immunogenicities of DNA vaccines expressing pol genes derived from B/C and A/E recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized pol genes derived from BYC and A/E subtypes of HIV-1 into mammalian expression vector pSV1.0. In vitro expression efficiencies of the two DNA vaccines were determined by Western blotting and their immunogeneeities were compared by i. m. immunizing female BALB/c mice. After immunization, mice splenocytes were isolated sterilely and IFN-γ based enzyme linked immunospot assay （ ELISPOT ） was employed to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blotting result showed both of the two DNA vaccines could be expressed at appreciable levels in vitro. Under the stimulation of Consensus B Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was （636 ± 178）SFCs/106 splenocytes; specific T cell frequency elicited by pSVCN-Pol was （468 ± 265 ） SFCs/106 splenocytes （ P = 0. 412 ）. Under the stimulation of HIV-1 AE2f Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was（ 1378 ± 611 ）SFCs/IO6 splenocytes; specific T cell frequency elicited by pSVCN-Pol was （ 713 ± 61 ） SFCs/106 splenocytes （ P = 0. 134 ） . Further analysis suggested pSVAE-Pol induced specific T cell responses mainly focused on Pol 1 peptide pool, while, in addition to induce Pol 1 specific T cell responses, pSVCN-Pol could also elicit T cell responses against consensus B Pol 2 peptide pool. Conclusion Although pSVAE-Pol was more immunogenic, pSVCN-Pol could induce T cell responses against broader epitope spectrum. Rational vaccine design may need combine them together.

关 键 词：HIV 疫苗 DNA T淋巴细胞 Pol蛋白 

分 类 号：R392.1[医药卫生—免疫学]