结缔组织生长因子的克隆及其真核表达载体的构建

目的结缔组织生长因子(CTGF)在肿瘤发生,胚胎发育、骨折愈合、细胞增殖和分化、血管形成等众多领域都有着重要的作用,对结缔组织生长因子进行克隆及构建其真核表达载体,对其在功能上的进一步研究及应用有积极意义.方法以含有鼠CTGF基因序列的EST克隆为模板,通过PCR的方法克隆出目的基因CTGF,再以分子克隆学的方法将CTGF克隆入真核表达载体pAdTrack-CMV,进一步以PCR、酶切、DNA测序的方法筛选出正确的真核表达质粒.并通过Western-Blotting的方法对质粒的表达功能进行研究.结果 PCR、酶切、DNA测序的方法均证实已经成功克隆获得序列正确的目的基因CTGF,并将CTGF克隆入真核表达载体pAdTrack-CMV.Western-Blotting的结果表明构建的表达CTGF的真核表达载体高表达CTGF蛋白质.结论成功克隆目的基因CTGF并构建其真核表达质粒,证实在蛋白质水平获得了表达.

CTGF gene cloning and the construction of its eukaryotic expression vector

Objective Connective tissue growth factor (CTGF) plays a very important role in a serial of fields such as tumorigenesis, embryonic development , bone fracture healing, cell proliferation and differentiation, and angiogenesis . There is a significance to deep into the functional research by CTGF gene cloning and the building of its eukaryotic expression vector. Methods we cloned the target gene CTGF from the murine gene bank by PCR, then subcloned this gene into the eukaryotic expression vector pAdTrcack-CMV,and picked up the correct expression eukaryotic expression plasmid DNA by PCR screening,enzyme digestion,DNA sequence screening.Finally we briefly explored the expressional function of recombination plasmid by Western blotting.Results We verified the correct target gene CTGF by PCR, enzyme digestion, DNA sequence screening and successfully cloned CTGF into the eukaryotic expression vector pAdTrcack-CMV. Finally, there is a high protein expression of CTGF from the eukaryotic expression plasmid tested by Western blotting.Conclusion We successfully amplified the target gene CTGF and established its eukaryotic expression plasmid. Further more we verified that there is a high protein expression by this recombination plasmid.