TERT多表位疫苗的制备及其免疫原性初步研究

Preparation and primary immunogenicity evaluation of multiepitope vaccine candidate from TERT

目的:制备端粒酶逆转录酶(TERT)的多表位(meTERT)肽疫苗和DNA疫苗,免疫小鼠,对其免疫原性进行初步研究。方法:用重叠延伸PCR将TERT的多个CD4+和CD8+T细胞表位串联连接,得meTERT的DNA片段;将该片段分别连接到真核表达载体pcDNA3.1及原核表达载体pGEX-4T中,制备DNA疫苗(PCT),并原核表达多表位肽疫苗(PCG);皮下/肌肉(50μg/100μg)免疫小鼠3次,取脾细胞,用ELISPOT法检测分泌IFN-γ和IL-4的细胞频率;用乳酸脱氢酶(LDH)释放法检测特异性脾细胞杀伤率(抗原刺激的SP2/0细胞为靶细胞)。结果:成功制备meTERT的PCT和PCG疫苗;动物实验表明:PCT+PCG联合免疫诱导的分泌IFN-γ的淋巴细胞数量显著高于其它各组(P<0.01);另外,PCT+PCG诱导的分泌IFN-γ的淋巴细胞数显著高于分泌IL-4的数量;PCT+PCG联合免疫诱导了显著的脾细胞杀伤活性(67.8%)。结论:含多个CD4+和CD8+T细胞表位的meTERT的DNA疫苗PCT和多表位肽疫苗PCG联合免疫能够诱导强烈的Th1和CTL细胞免疫应答。

Objective：To prepare the muhiepitope peptide or DNA vaccine candidates, and to assess the immunogenicity in immunized mice. Methods： Overlap extension PCR was used to obtain the meTERT DNA fragment from multiple CD4^＋ and CD8^＋ T cell epitopes of TERT. Then the fragment was subcloned into the eukaryotic expression vector pcDNA3.1 or prokaryotic expression vector pGEX-4T to construct the DNA vaccine （PCT） or to express peptide vaccine （PCG） in E. coli. Mice were immunized sc. ＇and mu. 3 time with 50 gg/100 μg, and splenocytes were tested for CTL activity by LDH （target cell： SP2/0 pulsed with the antigen） and the levels of IL-4 and IFN-γ-secreting lymphocytes were tested by ELISPOT. Results： PCT and PCG vaccine candidates were prepared successfully. Co-immunization with PCT ＋ PCG elicited significantly high frequency of IFN-y-secreting lymphocytes. The frequency of IFN-γ-secreting lymphocytes was higher remarkably than that of IL-4-secreting lymphocytes in mice immunized with PCT ＋ PCG. Moreover, PCT ＋ PCG induced high level of specific CTL activity. Conclusion：The DNA vaccine and peptide vaccine candidates （PCr ＋ PCG） of meTERT containing multiple CD4^＋ T cell epitopes and CD8^＋ T cell epitopes could induce strongly Thl and CTL cellular immune responses in mice.