河流型水牛热休克蛋白70的原核表达及其对精液抗冻性的影响  

Prokaryotic Expression of HSP70 and It's Effect on the Freezing Resistance of Semen in River Buffalo

作　　者：李芳芳 龙开旭[2] 俸祥仁 黄项生 潘堂峰 江明生 王国利[1] 邓祝新 蒋钦扬 郭亚芬[2] LI Fangfang;LONG Kaixu;FENG Xiangren;HUANG Xiangsheng;PAN Tangfeng;JIANG Mingsheng;WANG Guoli;DENG Zhuxin;JIANG Qinyang;GUO Yafen(Guangxi Work Station of Livestock&Poultry Breed Improvement,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530005,China;Guangxi Baipeng Livestock Breeding Station,Liuzhou 541502,China;New&High-tech Industrial Development Zone Animal Health Supervision Institute, Nanning 530007,China)

机构地区：[1]广西壮族自治区畜禽品种改良站,南宁530001 [2]广西大学动物科学技术学院,南宁530005 [3]广西百朋种畜场,柳州541502 [4]南宁高新技术产业开发区动物卫生监督所,南宁530007

出　　处：《中国畜牧兽医》2018年第9期2417-2424,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家农业科技成果转化资金重大项目(2014GB2E100283);广西水产畜牧科技推广应用项目(桂渔牧科(201633033;201528001));南宁市科学研究与技术开发计划项目(20172134-2);南宁市青秀区科学研究与技术开发计划项目(2017039)

摘　　要：试验旨在克隆河流型水牛热休克蛋白70(heat shock protein 70,HSP70)基因的CDS区序列,构建HSP70原核表达载体,诱导表达HSP70融合蛋白,进一步研究HSP70蛋白对水牛精液抗冻性的影响。以摩拉水牛精子基因组DNA为模板,采用PCR方法扩增HSP70基因CDS区序列,将PCR产物与pET30a质粒连接构建重组原核表达质粒,诱导表达HSP70融合蛋白,将获得的表达蛋白作为稀释液组分添加到水牛精液中制作冷冻精液并评定其活力。结果显示,试验成功扩增获得HSP70基因CDS区长为2 155bp的片段;SDS-PAGE及Western blotting分析显示,在70ku处出现一条明显条带,且表达产物可与相应的抗体发生反应;对添加有HSP70蛋白的水牛冷冻精液活力评定结果显示,终浓度为2、4、8mg/mL的HSP70能提高冷冻精液活力,且8mg/mL HSP70组冻精活力最高,但各组间差异均不显著(P>0.05)。综上所述,本试验成功表达了水牛HSP70蛋白,获得了高纯度、有活性的HSP70融合蛋白,外源添加HSP70蛋白可提高水牛冷冻精液的活力,为进一步研究水牛HSP70蛋白的抗冻性保护机理奠定了理论基础。The purpose of the research was to clone heat shock protein 70(HSP70)gene CDS region,construct the HSP70 prokaryotic expression vector,and induce the expression of HSP70 fusion protein to further study the effect of HSP70 on the freezing semen motility of river buffalo.The buffalo sperm genome DNA was extracted as a template,the HSP 70 gene CDS region was amplified by PCR,and the PCR products was connected with pET30a plasmid to construct recombinant prokaryotic expression plasmid and induce the expression of HSP70.The obtained HSP70 was added to the buffalo frozen semen and it’s motility was evaluated.The results showed that the CDS region of HSP 70 gene was amplified successfully with 2 155 bp in length.The SDS-PAGE and Western blotting analysis showed that the fusion protein had the molecular weight of 70 ku and could react with antibodies.The final concentration of 2,4 and 8 mg/mL HSP70 could improve the motilities of frozen semen,and 8 mg/mL HSP70 had the best effect on the motilities,but there were no significant differences among groups(P>0.05).In conclusion,the purified HSP70 obtained in this study had good quality,and could improve the frozen semen motility.The study laid a theoretical foundation for further studying the antifreeze protection mechanism of buffalo HSP70 protein.

关 键 词：河流型水牛 热休克蛋白70(HSP) 原核表达 精子活力 

分 类 号：Q786[生物学—分子生物学]