猪β-内啡肽基因克隆和重组质粒壳聚糖修饰分子包装体外表达研究  

Study on Cloning of Swine β-endorphin Gene and Expression in vitro of Recombinant Plasmid Packaged with Chitosan-modified Molecule

作　　者：白光明[1] 罗公明[1] 王晔[1] 李瑛[2] 高荣[1] 

机构地区：[1]四川大学生命科学学院,生物资源与生态环境教育部重点实验室,成都610064 [2]四川大学化学学院,成都610064

出　　处：《四川动物》2013年第1期44-50,共7页Sichuan Journal of Zoology

摘　　要：目的克隆猪β-内啡肽基因,研究其体外原核、真核表达,探索高效表达目的基因的新型纳米材料,为进一步研究β-内啡肽对动物免疫应答和应激调节等方面的作用奠定基础。方法利用基因延伸重叠PCR克隆β-内啡肽的基因cDNA,双酶切后插入VR1020、pGEX-4T-1,构建其真核、原核表达载体;以不同浓度的IPTG诱导表达重组融合蛋白,并作SDS-PAGE和RT-PCR分析目的基因原核表达情况;利用壳聚糖及其修饰分子制备纳米颗粒包装重组真核表达质粒,体外转染HEK293细胞,提取总RNA进行RT-PCR分析β-内啡肽基因在真核细胞的表达。结果成功克隆了猪β-内啡肽基因,并构建获得其原核和真核表达载体;不同浓度的IPTG诱导后,在29.7kDa的位置出现了新的蛋白条带,而原来的26.2kDa处的GST标签蛋白条带消失。说明目的基因已经和GST融合产生了新的融合蛋白;壳聚糖及其聚乙二醇和聚乙烯亚胺接枝修饰分子(mPEG-PEI-CS)纳米颗粒包裹VREP转染HEK293细胞,48h后收集细胞,RT-PCR和ELISA检测显示VREP能够在HEK293细胞中高效地转录表达β-内啡肽。结论本实验成功克隆了猪的β-内啡肽基因,并成功进行了原核及真核细胞表达分析,壳聚糖修饰分子纳米颗粒包装可高效表达目的基因,为进一步的动物实验奠定了基础。Objective In order to study the biological effect of β-endorphin on immune response and stress of animal, pigs β-endorphin gene is cloned in it＇s prokaryotic and eukaryotic expression is investigated in vitro, and we explored new nanomaterials for efficient expression of target genes. Methods The β-endorphin gene was cloned by the gene splicing of overlapping extension PCR technique. β-endorphin gene was then digested by double restriction enzyme and inserted into VR1020 and pGEX-4T-1 plasmid to respectively construct the recombinant eukaryotic and prokaryotic expression vector. And the fusion protein was induced to express by different concentrations of IPTG. RT-PCR and SDS-PAGE were applied to analyze the expression of target gene. Chitosan and its derivative modified with PEG and PEI were utilized to pack VREP plasmid into nanoparticles for transfection of HEK293 cell. The total RNA was isolated from the transfected cell on 48 h post transfection. Afterwards RT-PCR was used to evaluate the efficient expression of target gene in vitro. Results Pig β-endorphin gene was successfully cloned and the recombinant eukaryotic and prokaryotic plasmids were constructed. New 29.7 kDa protein was induced by IPTG in recombinant E. coli, while GST-tagged 26.2 kDa protein disappeared, which indicated that the target gene had successfully expressed a new fusion protein. The RT-PCR and ELISA results showed that the target gene was successfully transcribed in HEK293 cells, implying that VREP is able to express β-endorphin in HEK293 cells. Conclusion Pig β-endorphin gene was successfully cloned and expressed in eukaryote and prokaryote cell, and the modified chitosan nano-materials could significantly promote the expression of β-endorphin gene in vitro compared to the controls. These could facilitate the further research on its biological effect on animal and possible field application in the future.

关 键 词：猪β-内啡肽基因 克隆 高效表达 壳聚糖 细胞转染 

分 类 号：Q789[生物学—分子生物学]