金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 *

doi:10.13523/j.cb.2001070

中国生物工程杂志

2020, Vol. 40

Issue (7): 41-50 DOI: 10.13523/j.cb.2001070

技术与方法

金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 ^*

程旭¹,杨雨睛¹,吴赛男¹,侯勤龙^1,²,李咏梅^1,^2,^**(

),韩慧明^1,^2,^**(

)

1 北华大学医学院吉林 132013
2 北华大学医学院感染与免疫研究中心吉林 132013

Construction of DNA Vaccines of Staphylococcus aureus SarA, IcaA and Their Fusion Genes and Preliminary Study in Mouse Immune Response

CHENG Xu¹,YANG Yu-qing¹,WU Sai-nan¹,HOU Qin-long^1,²,LI Yong-mei^1,^2,^**(

),HAN Hui-ming^1,^2,^**(

)

1 Medical College of Beihua University, Jilin 132013, China
2 Center for Infection and Immunity Medical College of Beihua University, Jilin 132013, China

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摘要：

目的:探究SarA-AG、IcaA-AG及IcaA-SarA-AG(Azami Green用AG表示)融合基因的DNA疫苗及对小鼠免疫应答的影响。方法:以金黄色葡萄球菌基因组为模板,通过PCR等反应进行SarA-AG、IcaA-AG及IcaA-SarA-AG融合基因DNA疫苗的构建,将DNA疫苗转染至HeLa细胞,荧光显微镜下观察DNA疫苗的瞬时表达情况,将转染成功2周后的细胞进行基因组提取,PCR检测质粒在染色体上的整合情况。使用AG、SarA-AG(A组)、IcaA-AG(B组)及IcaA-SarA-AG(C组)4组免疫BALB/c小鼠,应用ELISA试剂盒检测小鼠血清中IgG抗体、IL-2、IL-4、IL-13、IFN-γ及TNF-α的分泌情况。结果:成功构建SarA-AG、IcaA-AG及IcaA-SarA-AG融合基因DNA疫苗,荧光显微镜下观察DNA疫苗转染情况,结果显示有绿色荧光。提取细胞基因组PCR检测结果显示未出现目的基因条带。免疫小鼠后,A、B、C组均能够诱导小鼠产生较高水平的IgG抗体。与空白组及空载体AG组相比,A、B组均能分泌较高水平的IL-2(P<0.001)、IFN-γ(P<0.001)、TNF-α(P<0.001)、IL-4(P<0. 01)及IL-13(P<0. 01),而C组TNF-α(P<0.05)、IL-4(P<0. 05)及IL-13(P<0. 05)分泌较少,但差异有统计学意义,细胞因子的分泌随着免疫次数的增加,差异显著增加。空白组及空载体AG组细胞因子无显著差异。结论:成功构建SarA-AG、IcaA-AG及IcaA-SarA-AG融合基因的DNA疫苗,且成功在真核细胞中表达。PCR验证结果证实了DNA疫苗的安全性。DNA疫苗免疫小鼠后可诱导体液免疫应答和以Th1细胞为主的细胞免疫应答,具有较好的应用前景。

关键词： DNA疫苗; 真核表达; 免疫效果

Abstract:

Objective: To investigate the effects of DNA vaccines on the fusion genes of SarA-Azami Green, IcaA-Azami Green, and IcaA-SarA-Azami Green (subsequently Azami Green was represented by AG) and their immune responses in mice.Method:Using the genome of S.aureus as a template, a series of reactions including SarA-AG, IcaA-AG, and IcaA-SarA-AG fusion DNA vaccines were constructed through a series of reactions such as PCR amplification, digestion, and ligation. After transfection into HeLa cells, the transient expression of DNA vaccine was observed under a fluorescent microscope. The cells after two weeks of successful transfection were subjected to genome extraction, and the integration of plasmids on chromosomes was detected by PCR. Recombinant plasmids were extracted using endotoxin-free plasmid extraction kits. BALB / c mice were immunized with AG, SarA-AG, IcaA-AG, and IcaA-SarA-AG. ELISA kits were used to detect mouse IgG antibodies, IL-2, IL-4, IL-13, IFN-γ and TNF-α secretion.Results: SarA-AG, IcaA-AG and IcaA-SarA-AG fusion gene DNA vaccines were successfully constructed. The transfection of the fusion gene DNA vaccine was observed under a fluorescence microscope, and the results showed green fluorescence. Genomic PCR results of the extracted cells showed that the target gene band did not appear. After immunizing mice, SarA-AG, IcaA-AG and IcaA-SarA-AG were able to induce mice to produce higher levels of IgG antibodies. Compared with the blank group and the empty vector AG group, the SarA-AG and IcaA-AG groups were able to secrete higher levels of IL-2 (P<0.001), IFN-γ (P<0.001), and TNF-α (P< 0.001), IL-4 (P<0. 01) and IL-13 (P<0. 01), while IcaA-SarA-AG group TNF-α (P<0.05), IL-4 (P<0. 05)) And IL-13 (P<0.05) were less secreted, but the difference was statistically significant. The secretion of cytokines increased significantly with the increase in the number of immunizations. There was no significant difference in cytokines between the blank group and the empty vector AG group.Conclusion: The DNA vaccines of SarA-AG, IcaA-AG and IcaA-SarA-AG fusion genes were successfully constructed and successfully expressed in eukaryotic cells. PCR verification results confirmed the safety of the DNA vaccine. DNA vaccine can induce humoral immune response and Th1 cell-based cellular immune response after immunizing mice, which has a good application prospect.

Key words: DNA vaccine Eukaryotic expression Immune effect

收稿日期: 2020-01-21 出版日期: 2020-08-13

ZTFLH:

Q812

基金资助: * 吉林省科技厅资助项目(20180101142J)

通讯作者: 李咏梅,韩慧明 E-mail: huiminghan@hotmail.com

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引用本文:

程旭,杨雨睛,吴赛男,侯勤龙,李咏梅,韩慧明. 金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 41-50.

CHENG Xu,YANG Yu-qing,WU Sai-nan,HOU Qin-long,LI Yong-mei,HAN Hui-ming. Construction of DNA Vaccines of Staphylococcus aureus SarA, IcaA and Their Fusion Genes and Preliminary Study in Mouse Immune Response. China Biotechnology, 2020, 40(7): 41-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001070 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I7/41

表1 引物序列

表2 SarA基因扩增体系

图1 SarA、IcaA、IcaA-SarA基因PCR结果图

图2 T载体重组质粒菌液PCR结果图

图3 T载体重组质粒双酶切鉴定结果图

图4 T载体重组质粒测序结果

图5 真核重组质粒双酶切鉴定结果图

图6 DNA疫苗细胞转染结果图

图7 PCR检测DNA疫苗安全性结果图

图8 小鼠血清IgG抗体水平检测

图9 小鼠血清细胞因子检测

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