ChIL-2-ChIL-4融合基因重组质粒遗传稳定性研究  

Genetic Stability Study of a Recombinant Gene Transfer Vector ChIL-2-ChIL-4

作　　者：郑龙龙 张黎 郝思源 郝飞飞 白瑞[1] 霍乃蕊[1] 郑明学[1] 谭凡 ZHENG Longlong;ZHANG Li;HAO Siyuan;HAO Feifei;BAI Rui;HUO Nairui;ZHENG Mingxue;TAN Fan(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学动物医学学院,太谷030801

出　　处：《激光生物学报》2021年第5期475-480,共6页Acta Laser Biology Sinica

基　　金：国家自然基金项目(31972647);山西省攻关项目(201703D221024-3);山西省科技成果转化项目(SXNKTG201802);山西省研究生创新项目(2021Y316);山西省博士毕业生、博士后研究人员来晋工作奖励资金科研项目(SXBYKY2021041)。

摘　　要：为研究大规模生产过程中DH5α/pUC57重组菌携带的表达鸡白细胞介素-2(ChIL-2)和白细胞介素-4(ChIL-4)融合基因质粒的遗传稳定性,探讨其作为鸡球虫活疫苗免疫佐剂的商业化批量生产的潜力,本文通过对重组质粒连续30代传代培养的方式,进行实验室遗传稳定性鉴定。研究结果显示:重组菌在含有氨苄青霉素(Amp+)的溶菌肉汤(LB)平板上传代30次均保持革兰氏阴性(G-),发酵葡萄糖及甘露醇并产生特异现象,但不发酵乳糖;重组菌与大肠杆菌16S rDNA片段的同源性均大于95%,各代重组质粒超螺旋比均大于90%,质粒丢失率均为0;重组质粒的表达产物具有刺激无特定病原体(SPF)雏鸡脾脏淋巴细胞的分化能力。结果表明,ChIL-2-ChIL-4融合基因重组菌和重组质粒30代内结构稳定、分离稳定,能够为扩大生产提供物质支持。The present study aims to investigate the genetic stability of the plasmid expressing chicken interleukin-2(ChIL-2)and chicken interleukin-4(ChIL-4)fusion genes carried by the DH5α/pUC57 recombinant strain during the large-scale production process,and to explore its potential as an immune adjuvant for live chicken coccidiosis vaccine in commercial batch production.The laboratory genetic stability identification of the recombinant DH5α/pUC57 was carried out by subculturing the recombinant plasmid for 30 consecutive generations.The results showed that after the recombinant strain being cultured for 30 consecutive generations on the lysic broth(LB)plates containing ampicillin(Amp+),the recombinant bacterias remained Gram-negative(G-),and could ferment glucose and mannitol to result in specific phenomena,whereas none fermented lactose was found.The homology of the 16S rDNA fragments between the recombinant bacteria and E.coli was greater than 95%,the supercoil ratio of the recombinant plasmids of each generation was greater than 90%,and the plasmid loss rate was 0.The expression product of the recombinant plasmid could stimulate the differentiation of spleen lymphocytes of SPF chicks.The above results indicate that there were essentially no changes in the structure of the 30 generations of continuous passages for the ChIL-2-ChIL-4 fusion gene recombinant bacteria and recombinant plasmids,and the plasmids can be isolated stably,which can provide material support for the expansion of production.

关 键 词：ChIL-2 ChIL-4 重组质粒 遗传稳定性 疫苗佐剂 

分 类 号：S855.9[农业科学—临床兽医学]