人透明质酸酶真核表达载体的构建及稳定转染CHO细胞系的建立

摘要

Objective To construct eukaryotic expression vector of human hyaluronidase (HAase) and transfect CHO cell line,and obtain the recombinant strain producing human HAase. Methods The cDNA encoding human HAase PH20 was synthesized and inserted into the eukaryotic expression vector MO2. After the identification by digestion and sequencing, the recombinant eukaryotic expression vector MO2/ph20, was then transfected into CHO cells by lipofectamine TM 2000. An engineering strain was finally selected out under the screening pressure 800 μg/mL G418. The expression of human HAase was identified by SDS-PAGE, and the biological activity of 4 positive cell clones were as well characterized in vitro with 3,5-dinitrosalicylic acid method.Results The eukaryotic expression vector was constructed successfully. The stable transfected CHO cell line was established. The HAase protein was expressed successfully and the molecular weight of recombinant human HAase was about 7.0×104. The activity of the recombinant human HAase of 4 positive cell clones were determined, and the activity of the 4th cell clone MO2/ph20-4 was more than 10 000 U/L. Conclusion The successful construction of the eukaryotic expression vector MO2/ph20 and establishment of the stable transfected CHO cells producing human HAase provided a good foundation for large scale production.%　　目的构建人透明质酸酶真核表达载体，并将其转染CHO细胞，建立稳定转染的CHO细胞系。方法根据已知的天然人透明质酸酶PH20的氨基酸序列，利用PCR技术，将其克隆至真核表达载体MO2中，得到表达质粒MO2/ph20。经酶切和测序鉴定后，用脂质体转染法转染CHO细胞，通过G418（800μg/mL）筛选，建立稳定转染的CHO细胞系，SDS-PAGE法检测重组人透明质酸酶的蛋白表达水平，利用3,5-二硝基水杨酸法检测筛选得到的4个阳性细胞克隆的生物活性。结果成功构建了MO2/ph20真核表达载体，并建立了稳定转染的CHO细胞系，成功地表达目的基因。重组人透明质酸酶表观相对分子质量约为70×103，4个阳性细胞克隆的酶活性均高于9000 U/L，其中MO2/ph20-4细胞株酶活性高达10000 U/L以上。结论本研究成功构建了能够正确表达人透明质酸酶基因的真核表达载体MO2/ph20，并获得了有生物活性的重组人透明质酸酶，该体系的建立为进一步规模化生产重组人透明质酸酶奠定了基础。