大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析  

Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages

作　　者：章志涛 李祥龙 孔令丽 罗雨昕 王冬英[1] ZHANG Zhitao;LI Xianglong;KONG Lingli;LUO Yuxin;WANG Dongying(Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics,Guangxi Key Laboratory of Animal Reproduction,Breeding and Disease Control,Key Laboratory of Prevention and Control for Animal Disease,College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西壮族自治区兽用生物制品工程研究中心,广西畜禽繁育与疾病防控重点实验室,广西高校动物疫病预防与控制重点实验室,南宁530004

出　　处：《中国畜牧兽医》2023年第3期1107-1117,共11页China Animal Husbandry & Veterinary Medicine

基　　金：广西自然科学基金(2019GXNSFAA245013);国家重点研发计划项目(2021YFD1100100、崇科FA2019006)。

摘　　要：【目的】克隆大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A(Ser/Thr protein phosphatases 2A,PP2A)基因并构建原核表达载体,获得原核表达蛋白,为探究PP 2 A基因功能及其在大片形吸虫的发育中的作用奠定基础。【方法】提取大片形吸虫成虫虫体组织总RNA,应用RT-PCR方法扩增PP 2 A基因的完整编码区序列,并以双酶切的方式连接pET-28a(+)载体;经酶切、测序鉴定后获得阳性质粒pET-28a-PP2A,将pET-28a-PP2A重组质粒转化大肠杆菌BL21(DE3)感受态细胞进行IPTG诱导表达,并对重组蛋白的诱导条件进行优化;通过SDS-PAGE及Western blotting检测大片形吸虫PP 2 A基因的表达效果;应用实时荧光定量PCR检测PP 2 A基因在大片形吸虫虫卵、囊蚴、6周龄童虫和成虫阶段中的表达情况。【结果】克隆的大片形吸虫PP 2 A基因编码区序列长1161 bp,编码386个氨基酸,分子式为C_(1980)H_(3105)N_(535)O_(566)S_(24),分子质量为44.23 ku。生物信息学分析结果显示,大片形吸虫PP 2 A基因编码的蛋白为PP2Ac,分子功能为信号转导(GO:0004871),生物学过程为蛋白去磷酸化(GO:0006470),细胞组分为细胞质(GO:0005829)。PP2A蛋白无信号肽,不存在跨膜结构域,为亲水性蛋白,共含有37个磷酸位点。SDS-PAGE和Western blotting结果显示,大片形吸虫PP2A在30℃、0.8 mmol/L IPTG诱导6 h时表达量最大,表达产物主要以包涵体的形式存在,且Western blotting检测结果显示,重组蛋白可被抗His标签识别;实时荧光定量PCR结果显示,PP 2 A基因在大片形吸虫6周龄童虫阶段表达量最高,极显著高于其他阶段(P<0.01);在囊蚴阶段的表达量最低,与虫卵阶段差异不显著(P>0.05)。【结论】本研究成功构建了大片形吸虫PP2A原核表达载体并获得相应蛋白,为研究PP 2 A基因在大片形吸虫生长发育中的功能奠定了基础。【Objective】The aim of this study was to clone the serine/threonine protein phosphatase 2A(Ser/Thr protein phosphatases 2A,PP2A)gene of Fasciola gigantica and construct a prokaryotic expression vector to obtain the prokaryotic expression protein,so as to lay a foundation for exploring the function of PP 2 A gene and its role in the development of Fasciola gigantica.【Method】Total RNA was extracted from adult trematodes,the complete coding region of PP 2 A gene was amplified by RT-PCR and ligated to pET-28a(+)vector by double restriction endonuclease digestion and sequencing,the positive plasmid was named as recombinant plasmid pET-28a-PP2A.The recombinant plasmid pET-28a-PP2A was transformed into E.coli expression strain BL21(DE3)competent cell for IPTG induction,and the induction conditions of the recombinant protein were optimized.The expression effect of PP 2 A gene of Fasciola gigantica was detected by SDS-PAGE and Western blotting.Real-time PCR was used to detect the expression of PP 2 A gene in eggs,metacercaria,6-week-old juvenile and adults of Fasciola gigantica.【Result】The coding region of PP 2 A gene of Fasciola gigantica was 1161 bp,encoding 386 amino acids.The molecular formula was C_(1980)H_(3105)N_(535)O_(566)S_(24)and the molecular weight was 44.23 ku.The results of bioinformatics analysis showed that the protein edited by PP 2 A gene was PP2Ac,the molecular function was signal transduction(GO:0004871),the biological process was protein dephosphorylation(GO:0006470),and the cell component was cytoplasm(GO:0005829).PP2A protein had no signal peptide and no transmembrane domain.It was a hydrophilic protein with 37 phosphate sites.The results of SDS-PAGE and Western blotting showed that the expression of PP2A in Fasciola gigantica was the highest at 30℃and 0.8 mmol/L IPTG inducing for 6 h.The expressed product was mainly in the form of inclusion body,and the recombinant protein could be recognized by anti-His tag by Western blotting detection.The results of Real-time PCR showed that the ex

关 键 词：大片形吸虫 丝氨酸/苏氨酸蛋白磷酸酶2A(PP2A) 原核表达 

分 类 号：S852.73[农业科学—基础兽医学]