人白细胞介素-10真核表达载体的构建及其在兔滑膜细胞中的表达

Construction of human interleukin-10 eukaryotic expression vector pcDNA4/HisMaxA-hIL-10 and its expression in rabbit synovial cells

目的构建人自细胞介素-10（hIL-10）高效真核表达载体，观察其在家兔滑膜细胞（RSCs）中表达。方法提取人外周血单个核细胞（PBMCs）总RNA作为模板，根据基因库（NM000572）中hIL-10全长开放读框设计特异性引物，反转录-聚合酶链反应（RT-PCR）一步法扩增hIL10mRNA全长开放读框，扩增产物定向克隆入真核表达载体pcDNA4／HisMaxA，并对克隆入真核表达载体的扩增产物进行酶切及DNA测序鉴定，构建的真核表达载体pcDNA4／Hi8MaxA-hIL-10经阳离子脂质体介导转染兔滑膜细胞RSCs。酶联免疫吸附试验（ELISA）检测转染后12、24、48、72h和7、14dRSCs培养上清中hIL-10水平。结果RT-PCR产物约0．54kh，克隆入真核表达载体DcDNA4／HisMaxA的扩增产物经酶切及DNA测序鉴定与读码框架序列无差别。转染后12h到第7天细胞培养上清检测出hIL-10，且水平显著性高于对照组（F=21．878，P〈0．01）。结论hIL-10真核高效表达载体pcDNA4／HisMaxA-hIL-10构建成功。

Objective To construct an efficient eukaryotic expression recombinant vector of human interleukin-10（hIL-10）, and observe its expression in rabbit synoviocytes（RSCs）. Methods Total RNA was extracted from peripheral blood mononuclear cells （PBMCs） of a patient with drug allergy. Specific primers for full-length open reading frames （ORFs） of hIL-10 were designed according to GeneBank （NM 000572 ）. With total RNA as the template, full-length ORFs of hIL-10 were amplified by reverse transcription polymerase chain reaction （RT-PCR）. RT-PCR products were digested by restrictive endonueleotidase, then inserted into plasmid peDNA4/HisMaxA. Both restrictive endonucleotidase analysis and DNA sequencing were carried out for inserts verification. RSCs were transfeeted with recombinant plasmid expression vector peDNA4 HisMaxAhiL10 by liposome-mediated gene transfer methods, then cultured in vitro. The supernatants were collected after transfeetion for 12 hours, 24 hours , 48 hours, 72 hours, 7 days, 14 days respectively for IL-10 measurement by enzyme linked immunosorbent assay （ELISA）. Results Full-length ORFs of hIL-10（0.54 kb） had been successfully cloned from PBMCs through RT-PCR. The inserts and insert location of pcDNA4 HisMaxA were in a right way verified by enzyme analysis and DNA sequencing. ELISA results showed that exogenous hIL-10 gene had expressed in the transfected RSCs from 12 hours to 7 days after transfeetion, and hIL-10 level of transfection group significantly higher than that of the control group. Conclusion peDNA4 HisMaxA- hiLl0, the hIL-10 efficient eukaryotie expression vectors, has been successfully constructed.