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目的构建和表达含信号肽的结核分枝杆菌8.4(MS)/人白细胞介素12(hIL-12)嵌合基因,并研究嵌合基因疫苗的免疫原性。方法克隆 MS/hIL12嵌合基因,导入真核表达载体 pCI-neo,构建成 MS/hIL12嵌合基因真核表达质粒,用限制性内切酶消化、聚合酶链反应(PCR)及 DNA 序列测定等多种分子生物学方法进行鉴定;重组嵌合质粒转染 COS-7细胞后,用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western blot)鉴定 MS/hIL12嵌合基因的表达情况。将 MS/hIL-12嵌合基因疫苗免疫 C57BL/6N 小鼠,脾细胞培养上清检测细胞因子水平;并按效、靶比例分别为100∶1、50∶1、10∶1进行细胞毒 T 淋巴细胞(CTL)杀伤检测。结果 MS/hIL12嵌合基因重组真核表达质粒构建成功;转染 COS-7细胞后,MS/hIL12嵌合基因在转录水平成功表达。MS/hIL-12嵌合基因疫苗组免疫小鼠脾细胞培养上清中γ干扰素(IFN-γ)和白细胞介素2(IL-2)含量分别为(1 521±48)ng/L 和(755±41)ng/L,MS 基因疫苗组分别为(820±50)ng/L 和(297±31)ng/L,BCG 组分别为(1 487±40)ng/L 和(767±50)ng/L,空载体组分别为(121±16)ng/L 和(62±10)ng/L,PBS 组分别为(48±16)ng/L 和(32±17)ng/L,上述结果显示 MS/hIL-12嵌合基因疫苗组 IFN-γ、IL-2分泌量增加,明显高于 MS 基因疫苗组及对照组(P<0.01),与 BCG 组相当(P>0.05);BCG 组免疫小鼠脾细胞培养上清中白细胞介素4(IL-4)的含量为(91±11)ng/L,明显高于其他各组(P<0.01)。效靶比为100∶1、50∶1、10∶1时,MS/hIL12嵌合基因疫苗组的 CTL 活性分别为77.5%、51.2%、30.3%,MS 基因疫苗组分别为56.2%、37.8%、11.5%,BCG 组分别为28.9%、21.4%、9.8%,MS/hIL-12嵌合基因疫苗组的CTL 活性高于 MS 基因疫苗组、BCG 组、空载体组和 PBS 组(P<0.01)。结论 hIL-12与 MS 构建成嵌合基因疫苗后,MS 基因疫苗的免疫原性得到很大提高。
Objective To construct and express the chimeric gene of Mycobacterium tuberculosis 8.4 (MS) / human interleukin 12 (hIL-12) containing signal peptide and investigate the immunogenicity of the chimeric gene vaccine. Methods The chimeric MS / hIL12 gene was cloned into the eukaryotic expression vector pCI-neo to construct the eukaryotic expression plasmid of MS / hIL12 chimeric gene. The restriction endonuclease digestion, polymerase chain reaction (PCR) and DNA sequencing The recombinant chimeric plasmid was transfected into COS-7 cells and the chimeric MS / hIL12 gene was identified by RT-PCR and Western blot The expression of the situation. C57BL / 6N mice were immunized with the chimeric MS / hIL-12 chimeric gene vaccine and cytokines levels were detected by spleen cell culture supernatants. Cytotoxicity was measured at 100: 1, 50: 1 and 10: 1 respectively Lymphocyte (CTL) killing assay. Results The recombinant eukaryotic expression plasmid of MS / hIL12 chimeric gene was constructed successfully. After transfection of COS-7 cells, the MS / hIL12 chimeric gene was successfully expressed at the transcriptional level. The levels of IFN-γ and IL-2 in splenocyte culture supernatant of mice immunized with MS / hIL-12 chimeric gene vaccine were (1 521 ± 48) ng / L and (755 ± 41) ng / L, (820 ± 50) ng / L and (297 ± 31) ng / L for MS gene vaccine group and (1 487 ± 40) ng / L and (121 ± 16) ng / L and (62 ± 10) ng / L for the empty vector group and (48 ± 16) ng / L and (32 ± 17) ng / L for the PBS group, respectively , The above results showed that the secretion of IFN-γ and IL-2 in the MS / hIL-12 chimeric gene vaccine group was significantly higher than that in the MS gene vaccine group and the control group (P <0.01) The level of interleukin-4 (IL-4) in the splenocyte culture supernatant of BCG group was (91 ± 11) ng / L, which was significantly higher than that of other groups (P <0.01). The CTL activities of the MS / hIL12 chimeric gene vaccine group were 77.5%, 51.2% and 30.3% respectively when the target ratios were 100: 1, 50: 1 and 10: 1, respectively, and 56.2% and 37.8% , 11.5% in BCG group and 28.9%, 21.4% and 9.8% in BCG group, respectively. The CTL activity of MS / hIL-12 chimeric gene vaccine group was higher than that of MS gene vaccine group, BCG group, ). Conclusion The immunogenicity of MS gene vaccine has been greatly improved after hIL-12 and MS were constructed into chimeric gene vaccines.