首页|猪TGF-βⅡ型受体胞外域的表达及生物活性验证

猪TGF-βⅡ型受体胞外域的表达及生物活性验证

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转化生长因子β1Ⅱ型受体(TGFⅡR)是TGF-β家族的主要受体.现有研究发现,TGF-β1 在家畜繁殖活动中起着负调控作用,因此我们提出通过重组TGFⅡR胞外域蛋白质竞争性结合体内TGF-β1 以改善家畜繁殖性能的新思路.首先,对猪TGFⅡR胞外域蛋白质编码序列进行密码子优化并进行人工合成,将合成的基因片段插入原核表达载体pET-32a(+)中.然后,将重组表达载体转入BL21(DE3)表达菌株中,并筛选合适的乳糖诱导浓度、诱导时间,以获得最高表达效率.再然后,对重组蛋白质进行纯化、复性及质谱鉴定.最后,通过体外细胞试验对重组蛋白质的生物活性进行测定.结果表明,在培养温度为 37℃、培养时间为 8h、乳糖诱导质量浓度为 2.0 g/L的条件下,可以诱导重组蛋白质的高表达,用 8 mol/L尿素对包涵体蛋白质进行溶解,再以生理盐水为透析液透析 24h后,可以获得纯度达 80%以上的重组猪TGFⅡR胞外域蛋白,用该重组蛋白质处理猪颗粒细胞后,可显著抑制TGF-β1 诱导的信号分子Smad3 的磷酸化水平.可以看出,本研究中表达的重组猪TGFⅡ R胞外域蛋白质具有较好的生物活性.研究结果可为进一步研究和开发有利于提高猪繁殖效率的产品奠定工作基础.
Expression of porcine TGF-β typeⅡ receptor in extracellular domain and validation of its biological activity
Transforming growth factor-β1 type Ⅱreceptor(TGFⅡR)is the main receptor of TGF-β family.Existing studies have shown that TGF-β1 plays a negative regulatory role in livestock reproduction activities.There-fore,we proposed a new idea to improve the reproductive performance of livestock by competitively binding TGF-β1 in vivo with recombinant TGFⅡR extracellular domain protein.In order to verify it,this study first optimized the codon of porcine TGFⅡ R extracellular domain and syn-thesized it artificially.The synthesized gene fragment was inserted into the prokaryotic expression vector pET-32a(+).Then,the recombinant expression vector was transferred into BL21(DE3)expression strain,and suitable lactose induction concentration and induction time were screened to obtain the highest expression efficiency.The recombinant protein was pu-rified,regenerated and identified by mass spectrometry.Finally,the bioactivity of the recombinant protein was determined by in vitro cell experiment.The results showed that the high expression of recombinant protein was induced at 37℃,6 h,2.0 g/L lactose,the inclusion body protein was dissolved by 8 mol/L urea,and the recombinant porcine TGFⅡR protein with a purity of more than 80%was obtained after dialysis with normal saline for 24 h.After treatment of porcine granulosa cells with the recombinant protein,the phosphorylation level of Smad3 induced by TGF-β1 was significantly inhibited,which indicated that the recombinant porcine TGFⅡR protein expressed in this study had good biological activity.In con-clusion,this study lays a foundation for further research and development of products suitable for improving the reproductive efficiency of pigs.

transforming growth factor-β1 typeⅡreceptor(TGFⅡR)prokaryotic expressionTGF-β1biologi-cal activity

赵蕾、李辉、覃水平、李碧侠、戴超辉、赵为民、付言峰、邓彦飞、程金花

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广西大学动物科学技术学院,广西 南宁 530005

农业农村部种养结合重点实验室/江苏省农业科学院畜牧研究所/江苏省农业种质资源保护与利用平台,江苏 南京 210014

广西壮族自治区环江毛南族自治县东兴镇农业技术推广站,广西环江 547117

转化生长因子β1Ⅱ型受体(TGFⅡR) 原核表达 TGF-β1 生物活性

江苏农业科技自主创新基金项目江苏省种业振兴揭榜挂帅项目国家自然科学基金项目江苏省自然科学基金项目财政部和农业农村部国家现代农业产业技术体系项目

CX223197JBGS202110232272883BK20211140CARS-PIG-35

2024

10.3969/j.issn.1000-4440.2024.07.013

江苏农业学报
江苏省农业科学院

江苏农业学报

CSTPCD北大核心
影响因子：1.093
ISSN：1000-4440
年,卷(期)：2024.40(7)