摘要
弓形虫是一种严重危害公共卫生安全的人兽共患寄生原虫。前期研究发现,相对于弓形虫Ⅰ型株(RH株),ChineseⅠ型株(YZ-2株)GRA7蛋白因缺失1个谷氨酸而形成新的抗原表位,但该表位对GRA7蛋白免疫学功能的影响尚不清楚。分别对弓形虫RH和YZ-2株GRA7基因进行PCR扩增,构建原核表达载体RH-pET30a(+)-GRA7和YZ-2-pET30a(+)-GRA7,并进行体外诱导表达;Western blot分析2种重组蛋白的抗原性及交叉反应原性。结果表明:GRA7基因PCR产物大小630~633 bp;体外诱导表达产物大小约37 ku;2种重组蛋白均能被抗His标签单克隆抗体、GRA7重组蛋白制备的鼠多克隆抗体以及弓形虫速殖子可溶性抗原制备的鼠多克隆抗体所识别;同时,用重组蛋白制备的多克隆抗体可交叉识别2种基因型虫体内的GRA7天然蛋白。Ⅰ型和ChineseⅠ型虫株的GRA7蛋白具有良好的交叉反应原性,该结果为进一步探索弓形虫GRA7蛋白的功能及其抗感染免疫保护作用的研究奠定基础。
Toxoplasma gondii is a zoonotic parasitic protozoan that seriously threatens public health safety. In our previous study, compared to type I strain(RH strain), GRA7 protein of Chinese I genotype strain(YZ-2 isolate) was lack of a glutamic acid, which formed a new epitope. However, the effects of this epitope on the immunological function of GRA7 protein is still unclear. Therefore, PCR was used to amplify the GRA7 gene fragments of RH strain and YZ-2 strain, respectively. The recombinant plasmids RH-pET30 a(+)-GRA7 and YZ-2-pET30 a(+)-GRA7 were constructed for inducing expression. The antigenicity and cross-immunogenicity of the recombinant proteins GRA7 of the two genotypic strains were analyzed by Western blot, respectively. Results showed that the PCR product size of GRA7 gene was 630-633 bp. After induction expression, the size of fusion protein was about 37 ku. Western blot analysis suggested that the two recombinant proteins can be specifically recognized by anti-His tag monoclonal antibody and mouse polyclonal antibodies prepared by GRA7 recombinant antigens and the soluble antigen of RH strain;simultaneously,the polyclonal antibodies prepared with the recombinant proteins can cross-recognize the GRA7 natural protein in the two genotypic strains.The GRA7 proteins of type I and type Chinese I strains had good antigenicity and cross-immunogenicity.This study laid the foundation for further research on the function and anti-infection immune protection of GRA7 protein of T.gondii.
作者
朱世范
李慧中
赵振兴
邱金姝
张会
徐康智
陈凌志
朱梦月
刘丹丹
许金俊
潘志明
陶建平
候照峰
ZHU Shifan;LI Huizhong;ZHAO Zhenxing;QIU Jinshu;ZHANG Hui;XU Kangzhi;CHEN Lingzhi;ZHU Mengyue;LIU Dandan;XU Jinjun;PAN Zhiming;TAO Jianping;HOU Zhaofeng(Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses/Jiangsu Key Laboratory of Zoonosis/College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2021年第6期60-66,共7页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
国家自然科学基金资助项目(32002303)
江苏省基础研究计划项目(BK20190885)
中国博士后科学基金项目(2020M671615)
江苏省人兽共患病学重点实验室开放课题(R1912)
扬州大学大学生科创基金项目(X20190681、X20210701)
江苏省高校优势学科建设工程项目(PAPD)。
