重组沙门菌C78-1△crp（pEGFP-WAP-HNP-3）的构建及生物学特性研究  

Construction and biological characteristics of the recombinant attenuated Salmonella choleraesuis C78-1△crp （pEGFP-WAP-HNP-3）

作　　者：陈慧敏[1,2] 李银聚[1] 张鹏[1] 陈松彪[1] 张春杰[1] 程相朝[1] 

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室,洛阳471003 [2]潢川县动物卫生监督所,信阳465150

出　　处：《中华微生物学和免疫学杂志》2016年第11期801-806,共6页Chinese Journal of Microbiology and Immunology

基　　金：河南省科技攻关项目（132102110117）

摘　　要：目的：构建大鼠乳清酸蛋白（WAP）启动子调控下的人防御素3（HNP-3）的重组减毒猪霍乱沙门菌C78-1△crp（pEGFP-WAP-HNP-3）,并对其生物学特性进行研究。方法将质粒pEGFP-WAP-HNP-3电转到减毒猪霍乱沙门菌C78-1△crp感受态,构建重组沙门菌C78-1△crp（ pEGFP-WAP-HNP-3）,并进一步研究其生物学特性。结果 PCR和测序结果表明C78-1△crp（pEGFP-WAP-HNP-3）构建成功。进行生物学特性分析表明,重组菌与亲本株C78-1相比失去了利用麦芽糖、鼠李糖等的能力,生长速度显著减慢,能够稳定遗传所携带的HNP-3基因,重组菌株的毒力约是亲本株C78-1的1/300,且重组菌注射到兔乳腺后的分泌乳中能够检测到HNP-3融合蛋白条带。结论重组菌C78-1△crp（pEGFP-WAP-HNP-3）构建成功,并能够分泌HNP-3融合蛋白,为进一步研究重组菌的功能提供基础资料。Objective To construct a recombinant attenuated Salmonella choleraesuis C78-1Δcrp （pEGFP-WAP-HNP-3） expressing the human defensin 3 （HNP-3） under the regulation of whey acidic pro-tein （WAP） promoter and to analyze its biological characteristics. Methods The recombinant plasmid pEGFP-WAP-HNP-3 was transformed into an attenuated Salmonella choleraesuis C78-1Δcrp by electropora-tion. Biological characteristics of the constructed recombinant attenuated Salmonella choleraesuis C78-1Δcrp （pEGFP-WAP-HNP-3） were studied. Results PCR and sequencing analysis showed that the C78-1Δcrp （pEGFP-WAP-HNP-3） was successfully constructed. Compared with the parent strain, the recombinant strain lost its ability to utilize maltose, rhamnose, etc. Significantly decreased growth rate of the C78-1Δcrp （pEGFP-WAP-HNP-3） contributed to the stable inheritance of the carried HNP-3 fusion genes. The viru-lence of the parent strain C78-1 was about 300 times that of the C78-1Δcrp （ pEGFP-WAP-HNP-3 ） . The HNP-3 fusion protein could be detected in the rabbit milk after injection of the recombinant C78-1Δcrp （ pEGFP-WAP-HNP-3） into the rabbit mammary gland. Conclusion The recombinant Salmonella cholerae-suis strain of C78-1Δcrp （ pEGFP-WAP-HNP-3 ） was constructed successfully and capable of secreting HNP-3 fusion protein. This study paved the way for further researches on the functions of recombinant bacteria.

关 键 词：减毒沙门菌C78—1△crp 人防御素3 重组沙门菌 生物学特性 

分 类 号：R378[医药卫生—病原生物学]