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。
关键词：ChIL-2；ChIL-4；重组质粒；遗传稳定性；疫苗佐剂
中图分类号：S855.9                          文献标志码：ADOI：10.3969/j.issn.1007-7146.2021.05.013

Abstract: 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.
key words: ChIL-2; ChIL-4; recombinant plasmid; genetic stability; vaccine adjuvant
（Acta Laser Biology Sinica, 2021, 30(5): 475-480）