含信号肽的结核分枝杆菌8.4/白细胞介素12嵌合基因真核表达质粒的构建、表达及免疫原性研究

Construction,expression and immunogenicity study of a chimeric MS/hIL-12 eukaryotic expression plasmid

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 a chimeric Mtb8.4 with signal peptide （MS）/ hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines. Methods The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction（PCR） and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 （pMSI） recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-γ cells were transfected with pMSI constructs by cationic liposom. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture superuatnant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte （CTL） induction. Results The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-7 and IL-2 titers were （1 521±48） ng/L and （755±41 ） ng/L in MS/hIL-12 chimeric gene vaccine group, （820±50） ng/L and （297±31 ） ng/L in MS gene vaccine group, （1 487±40） ng/L and（767±50） ng/L in BCG group, （ 121±16） ng/L and （62±10） ng/L in vacant vector group, and （48±16） ng/L and （32±17） ng/L in PBS group respectively. The levels of IFN-3, and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group （ P 〈 0. 01 ） and was similar to the BCG group （ P 〉 0.05）. The level of IL-4 in BCG group [ （91±11） ng/L] increased significantly as compared to other groups （ P 〈 0. 01 ）. When effector-cell-to-target-cell ratio （ E： T ratio ） were 100 ： 1,50： 1, and 10： 1 respectively, the CTL activity was 77. 5% ,51.2%, 30. 3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups （ P 〈 0.01 ）. Conclusion When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.