摘要
【目的】构建广西巴马小型猪结缔组织生长因子(CTGF)基因真核表达载体并鉴定其活性,为深入研究CTGF在癌症发生中的作用及其分子机制提供参考,也为构建CTGF转基因广西巴马小型猪及研发基于CTGF的癌症基因治疗策略提供理论依据。【方法】利用RT-PCR从广西巴马小型猪肝脏组织中克隆CTGF基因,并将CTGF基因亚克隆到p DsRed-N1载体上,然后通过脂质体转染小鼠Hep3B细胞,转染48 h后于荧光显微镜下观察细胞是否表达红色荧光蛋白,同时利用在线生物信息学软件对其理化性质进行分析。【结果】成功克隆获得广西巴马小型猪CTGF基因编码区(CDS)序列,序列全长1050 bp,共编码349个氨基酸,与NCBI上已公布普通猪参考序列的同源性为99.5%,有5处碱基突变,且均为同义突变。将广西巴马小型猪CTGF基因亚克隆到pDsRed-N1载体上成功构建了阅读框架正确的广西巴马小型猪CTGF基因真核表达载体,转染小鼠Hep3B细胞48 h后有红色荧光蛋白表达。广西巴马小型猪CTGF蛋白具有较强的亲水性,其二级结构中α-螺旋、β-折叠、延伸链和无规则卷曲各占13.18%、5.73%、17.48%和63.61%。【结论】广西巴马小型猪CTGF基因在进化过程中的保守性非常稳定,通过构建的真核表达载体能转染小鼠Hep3B细胞并成功表达,可用于生产转基因广西巴马小型猪及研发与CTGF基因有关的疾病模型。
【Objective】The connective tissue growth factor(CTGF)gene eukaryotic expression vector of Guangxi Bama miniature pigs was constructed and its activity was identified.It would provide reference for study in function of CTGF in cancer and its molecular mechanism,and also for developing CTGF transgenic Guangix Bama miniature pig and cancer genes treatment strategy based on CTGF.【Method】The CTGF gene was cloned by RT-PCR from liver of Guangxi Bama miniature pig.CTGF gene was subcloned into pDsRed-N1 vector,and was transfected into rat Hep3B cells by lipofection.After 48 h,the cells were observed under a fluorescence microscope to find out whether it expressed red fluorescent protein.At the same time,online bioinformatics software was used to analyze its physical and chemical properties.【Result】The gene encoding region(CDS)sequence of CTGF gene of Guangxi Bama miniature pig was successfully cloned.The sequence length was 1050 bp,encoding 349 amino acids.The homology with the ordinary pig reference sequence on NCBI was 99.5%.There were five base mutations,all of which were synonymous mutations.The eukaryotic expression vector of CTGF gene of Guangxi Bama miniature pigs with correct reading frame was successfully constructed by subcloning CTGF gene into pDsRed-N1 vector.After transfection,Hep3B cells expressed red fluorescent protein after 48 h.The CTGF protein of Guangxi Bama miniature pig was highly hydrophilic.The α-helix,β-sheet,extended chain and random coil of CTGF protein account for 13.18%,5.73%,17.48% and 63.61% in the secondary structure,respectively.【Conclusion】The CTGF gene of Guangxi Bama miniature pig is very stable during the evolution process,and it transfects mouse Hep3B cell through the constructed eukaryotic expression vector and successfully expresses.CTGF gene of Guangxi Bama miniature pigs can be used to produce transgenic Guangxi Bama miniature pig,and develop disease models associated with the CTGF gene.
作者
黄叶
吴延军
朱思燃
奉玲丽
瞿秋红
江雨航
夏琴
崔悦悦
莫家远
兰干球
HUANG Ye;WU Yan-jun;ZHU Si-ran;FEN Ling-li;QU Qiu-hong;JIANG Yu-hang;XIA Qin;CUI Yue-yue;MO Jia-yuan;LAN Gan-qiu(College of Animal Science & Technology,Guangxi University,Nanning 530004,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2019年第3期468-476,共9页
Journal of Southern Agriculture
基金
广西创新驱动发展专项项目(桂科AA17204029)
