水牛瘦素基因的真核表达及生物学特性分析

Eukaryotic Expression and Biological Characterization of leptin Gene in Buffalo

为研究水牛瘦素(Leptin)的功能,构建leptin基因真核表达载体EGFP-leptin,检测leptin在NIH/3T3细胞中的表达并分析其生物学特性。根据水牛leptin基因序列设计引物,并在上下游引物分别添加HindⅢ和BamHⅠ酶切位点,以pMD18-T-leptin载体为模板,PCR扩增leptin基因,并亚克隆至载体pEGFP-N1,以双酶切和DNA测序鉴定EGFP-leptin重组质粒;然后采用脂质体2000将重组质粒转染到NIH/3T3细胞,经G418筛选,获得稳定转染的细胞株,RT-PCR及Western blot检测EGFP-Leptin融合蛋白表达。动物试验分3组,处理组从小鼠尾静脉注入重组质粒1μg/只,对照组和禁食组注射等体积生理盐水,3 d后称其体质量,采用ELISA法测定小鼠血清中Leptin水平。结果显示,成功构建真核表达载体EGFP-leptin,转染NIH/3T3细胞后出现绿色荧光;RT-PCR和Western blot进一步证实了Leptin表达。动物试验显示,处理组和禁食组小鼠体质量下降速率高于对照组,差异显著(P<0.05),处理组血清Leptin水平高于对照组,差异显著(P<0.05)。以上结果表明,EGFP-leptin能够在NIH/3T3细胞中稳定表达,产生的融合蛋白具有良好的生物学活性。

To study the function of Leptin in buffalo,a eukaryotic expression vector EGFP‑leptin was constructed,the expression of leptin in NIH/3T3 cells was detected and its biological activity was determined.The primers were designed based on the sequence of buffalo leptin gene in GenBank,and the restrictive endonuclease sequences of both HindⅢand BamHⅠwere added to the forward and the reverse primers respectively.By means of PCR,the leptin gene was amplified using pMD18‑T‑leptin vector as a template,and subcloned to the pEGFP‑N1 vector to construct the EGFP‑leptin vector,which was identified by double restrictive endonuclease digestion and DNA sequencing.Then the recombinant plasmid was transfected into NIH/3T3 cells by liposome 2000,and stable transfected cell lines were screened using G418.The expression of EGFP‑Leptin fusion protein was verified by RT‑PCR and Western blot analyses.Ine three groups of animal tests,EGFP‑leptin plasmid(1μg in 100μL saline)was administrated via tail intravenous injection in the treatment group,1μg each,and the fasting group and control group were administrated with the same volume of saline in the same way.All mice were weighed and serum Leptin levels were determined by ELISA after three days.The results showed that the EGFP‑leptin was successfully constructed,green fluorescence was observed in NIH/3T3 cells transfected with the recombinant plasmid,and the expression of leptin was further verified through RT‑PCR and Western blot assays.Animal tests showed that the weight loss ratio in the treatment group and fasting group was significantly faster than in the control group(P<0.05),and the serum Leptin level in the treatment group was significantly higher than in the control group(P<0.05).In conclusion,EGFP‑leptin was stably expressed in NIH/3T3 cells and the fusion protein was functional biologically.