双表达去势DNA疫苗的构建及其稳定性研究

doi:10.16431/j.cnki.1671-7236.2021.05.026

摘要/Abstract

摘要： 试验旨在构建一种更安全有效的新型免疫去势DNA疫苗，通过选取下丘脑-垂体-性腺轴（hypot halamic-pituitary-gonadal axis，HPG）的上游调控基因吻素1（KISS1）和促性腺激素释放激素（GnRH）作为靶标，借助2A肽的自剪切功能，引入平衡致死系统代替抗性基因筛选流程，成功将GnRH和KISS1转入非抗性筛选质粒pVAX-asd中，酶切验证和测序对比验证目的基因的插入方向和序列完全正确。重组质粒转染HeLa细胞，反转录后扩增目的基因结果显示，重组质粒在真核细胞内能够正常转录，保证重组质粒在导入机体后能够正常表达，从而引起特异性免疫反应。将构建成功的质粒转入减毒的猪霍乱沙门氏菌C500中，获得可直接口服免疫的活载体疫苗，酶切和测序结果表明，双表达重组质粒成功导入工程菌中。将活菌疫苗在体外连续传代50次，选取0、2、5、10、20、30、40、50代的菌株进行稳定性研究，生长曲线检测结果表明，工程菌在体外连续传代50次的过程中，其生长特性无明显变化，且与减毒猪霍乱沙门氏菌C500的生长特性一致，未因携带质粒发生明显变化；同时将各代菌液扩增沙门氏菌标志基因（invA）和毒力基因（crp），结果表明多次传代后工程菌仍然具有沙门氏菌的特性，其减毒特性也无变化。各代菌液质粒酶切验证显示，多次传代并不影响质粒的稳定性，重组双表达质粒能够在沙门氏菌C500中维持正常拷贝功能。综上所述，该重组质粒和工程菌疫苗均具有良好的稳定性，可直接应用于动物免疫去势的研究。

关键词: DNA疫苗; KISS1; GnRH; 2A肽; 免疫去势; 稳定性

Abstract: The aim of current study was to construct a safer and more effective new type of immune castration DNA vaccine.With the help of the self-cleavage function of the 2A peptide and introducting of a balanced lethal system instead the resistance gene screening process,the hypot halamic-pituitary-gonadal axis (HPG) upstream regulatory gene kissin 1 (KISS1) and hormone-releasing hormone (GnRH) being selected as the target were successfully transferred into the non-resistant screening plasmid pVAX-asd.Enzyme digestion verification and sequencing comparison verification on the insertion direction and sequence of the target gene were completely correct.When recombinant plasmid being transfected into HeLa cells,the results of amplification of the target gene after reverse transcription showed that the recombinant plasmid could be transcribed normally in eukaryotic cells,ensuring that the recombinant plasmid could be expressed normally after being introduced into the body,thereby causing a specific immune response.The successfully constructed plasmid was transformed into the attenuated Salmonella choleraesuis C500 to obtain a live vector vaccine that could be directly orally immunized.The results of enzyme digestion and sequencing showed that the co-expression recombinant plasmid was successfully introduced into the engineered bacteria.The live bacteria vaccine was continuously passaged in vitro for 50 times,and strains of 0,2,5,10,20,30,40,and 50 generations were selected for stability study.The growth curve test results showed that there was no significant change in its growth characteristics when the engineered bacteria were continuously passaged in vitro for 50 times,and it was consistent with the growth characteristics of attenuated Salmonella choleraesuis C500.There also was no significant changes were found when it carrying plasmids.At the same time,Salmonella marker genes (invA) and virulence genes (crp) was used to amplified with each generation of bacterial fluid,the results showed that the engineered bacteria still had the characteristics of Salmonella after multiple passages,and their attenuation characteristics had not changed.Direct enzyme digestion verification of bacterial liquid with various generations showed that multiple passages did not affect the stability of the plasmid,and the recombinant co-expression plasmid could maintain normal copy function in Salmonella C500.In summary,both the recombinant plasmid and the engineered bacterial vaccine had good stability,which could be directly applied to the study of animal immunocastration.

Key words: DNA vaccine; KISS1; GnRH; 2A peptide; immune castration; stability

中图分类号:

S852.4

许学林, 杨波, 荆焕松, 胡映红, 王风华, 孙培皓, 熊家军. 双表达去势DNA疫苗的构建及其稳定性研究[J]. 中国畜牧兽医, 2021, 48(5): 1745-1754.

XU Xuelin, YANG Bo, JING Huansong, HU Yinghong, WANG Fenghua, SUN Peihao, XIONG Jiajun. Construction and Stability Study of Co-expression Castration DNA Vaccine[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1745-1754.

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