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

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2 155 bp的片段；SDS-PAGE及Western blotting分析显示，在70 ku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、8 mg/mL的HSP70能提高冷冻精液活力，且8 mg/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。

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

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 HSP70 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 HSP70 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.