牦牛BNBD4组织分布及其蛋白特性的研究  

Study on tissues distribution and protein characteristics of BNBD4 in Bos grunniens

作　　者：郑姚 李娟[1] 王利[1] 陈希文[2] ZHENG Yao;LI Juan;WANG Li;CHEN Xi-wen(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education and Sichuan Province,Southwest Minzu University Chengdu 610041,China;Animal Disease Prevention and Control and Healthy Breeding Engineering Technology Research Center,Mianyang Normal University,Mianyang 621000,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部和四川省重点实验室,四川成都610041 [2]绵阳师范学院动物疫病防控与健康养殖工程技术研究中心,四川绵阳621000

出　　处：《中国预防兽医学报》2020年第10期1045-1050,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：西南民族大学中央高校基本科研业务费专项资金项目资助(2020NYB30);四川省留学人员科技活动择优资助项目(2019);阿坝州科技局项目(18YYJSYJ0015)。

摘　　要：为探究牦牛中性粒细胞β防御素4(BNBD4)在牦牛组织中的分布情况以及BNBD4的蛋白特性,本研究采用RT-PCR技术扩增获得牦牛BNBD4基因序列,利用生物信息学软件预测其编码蛋白的结构与功能,结果显示,牦牛BNBD4基因开放阅读框为195 bp,编码64个氨基酸,其序列与黄牛和水牛的相似性分别为96.4%和87.7%,其在同属中具有高度的保守性。蛋白功能与结构预测结果显示,该蛋白属于分泌型阳离子β防御素,其可与病原体的磷脂膜结合,从而破坏病原体细胞膜的完整性。通过荧光定量PCR检测BNBD4在牦牛5种组织中的转录水平,结果显示该基因在牦牛肾脏的转录水平极显著高于心、肝、脾和肺(p<0.01),其次该基因在肝脏中的转录水平相对较高。构建重组表达质粒pET32a-BNBD4并诱导表达,对表达蛋白进行SDS-PAGE和western blot鉴定分析,结果显示,BNBD4重组蛋白在大肠杆菌中以包涵体形式表达,重组蛋白约为23 ku。进一步将pET32a-BNBD4/BL21(DE3)经IPTG诱导后于LB平板过夜培养,观察各组菌落数,分析BNBD4对E.coli BL21(DE3)的毒性作用,结果显示,重组蛋白BNBD4对宿主大肠杆菌的生长具有一定的抑制作用。本研究检测分析了牦牛BNBD4的组织分布,并首次原核表达了牦牛BNBD4蛋白,初步鉴定了其特性,为探究BNBD4基因在牦牛中的功能奠定了基础。In this study,to explore tissues distribution of Bos grunniens neutrophil beta-defensin 4(BNBD4)and the characteristics of BNBD4 protein,reverse transcription(RT-PCR)was used to amplify the BNBD4 gene of Bos grunniens.The structure and function of the BNBD4 protein were predicted byrelevant bioinformatics software.The results showed that the open reading frame of the BNBD4 was 195 bp,which encoded 64 amino acids.The CDS sequence of BNBD4 showed 96.4%and 87.7%homology with that of Bos taurus and Bubalusbubalis,respectively and it was highly conserved among its genus.The predicted results about function and structure of the protein showed that the protein belongs to secretory cationic beta defensin,which can bind to the phospholipid membrane of pathogen,thus destroying the integrity of pathogen cell membrane.The transcription of BNBD4 gene in different tissues was detected by real-time PCR.The results of real-time PCR showed that the transcription level of BNBD4 in kidney was significantly higher than that in other(heart,liver,spleen and lung)tissues(p<0.01),followed by liver in Bos grunniens.The BNBD4 gene ORF was cloned into the prokaryotic expression plasmid pET-32a,and the obtained recombinant plasmid pET32a-BNBD4 was then expressed and identified by SDS-PAGE and western blot.BNBD4 was expressed in the form of inclusion bodies in E.coli and the recombinant protein was about 23 ku.The toxicity test results showed that rBNBD4 affected the growth of E.coli.This research first provides basic data for further exploring the function of BNBD4 in the Bos grunniens immune system.

关 键 词：BNBD4 牦牛 组织表达 原核表达 

分 类 号：S852.4[农业科学—基础兽医学]