Ⅱ型胶原蛋白基因cDNA序列分析及其真核表达载体的构建

Sequence analysis of type Ⅱ collagen protein cDNA and construction of its’eukaryotic expression vector

目的构建人Ⅱ型胶原蛋白(hCⅡ)基因真核表达载体PcDNA3.1(+)/hCⅡ。方法提取患者关节软骨组织总RNA,应用RT-PCR方法扩增出CⅡ基因片段,测序鉴定正确后,插入真核表达载体PcDNA3.1(+)中构建hCⅡ基因真核表达载体PcDNA3.1(+)/hCⅡ,并进行PCR鉴定、酶切鉴定和序列分析。结果 PCR鉴定、酶切鉴定和序列分析表明CⅡ成功插入表达载体PcDNA3.1(+)。结论本研究成功构建了hCⅡ基因真核表达载体PcDNA3.1(+)/hCⅡ。

Objective To construct an eukaryotic expressing vector PcDNA3.1 （ ＋ ）/gene of type Ⅱ collagen （C Ⅱ ） protein. Methods Total RNA from articular cartilage cells in patients with rheumatoid arthritis was extracted and the CⅡ gene fragments were amplified by RT-PCR method. The sequencing was identified, and inserted into the eukaryotic expression vector PcDNA3.1 （ ＋ ） to construct the human C II gene eukaryotic expression vector PcDNA3.1 （ ＋ ）/hC Ⅱ. And construction of expression vector was identified by PCR, restriction endonuclease digestion and sequence analysis. Result C Ⅱ gene was successfully inserted into the expression vector PcDNA3.1 （ ＋ ） by PCR, restriction endonuclease digestion and sequence analysis. Conclusion This study has successfully constructed the eukaryotic expression vector PcD- NA3.1 （ ＋ ）/hC Ⅱ.