【摘要】 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。更多还原

【Abstract】 Objective To construct and express a chimeric Mth8.4 with signal peptide (MS)/ hIL12 eukaryotic expression plasmid,and to study the immunogenieity 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-7 cells were transfeeted 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 supernatnant 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-γ 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-γ 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.更多还原