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《Chinese Journal of Blood Transfusion》 2007-06
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In vitro expression and purification of heavy chain and β_2m of soluble HLA-G1

XIA Rong1,QIU Huiying2,CAO Qiong3,et al.1.Department of Blood Transfusion,Hushan Hospital,Shanghai 200040,China;2.Department of Blood Transfusion, Nanfang Hospital;3.Department of Hematology,Changhai Hospital  
Objective To obtain purified heavy chain molecule and β-2microglobulin(β2m) light chain of soluble HLA-G1 protein.Methods RT-PCR was used to amplify sHLA-G heavy chain and β2m light chain genes from total RNA obtained from human placenta tissue by TRIzol.After DNA sequencing and restrictive enzyme digestion,the DNA fragments of sHLA-G heavy chain were purified from the cloning plasmids digested by BamHⅠand EcoRⅠ,while β2m were digested by NdeⅠ and HindⅢ.Then the fragments were inserted into plasmid pET28a(+),digested by the same enzyme,to construct recombinant expression.Expressions of sHLA-G1 heavy chain and β2m light chain by plasmids pET28a(+)/sHLA-G1 and pET28a(+)/β2m,were induced by ITPG in E.coli BL21.The sHLA-G1 heavy chain was purified by Co-affinity chromatography,and β2m purified by CM-FF weak cationic column.The purification and expression of the protein was identified by SDS-PAGE and Westernblot.Results The sHLA-G1 and β2m gene were homologous with the published gene sequences of sHLA-G1.The expression rate of sHLA-G1 heavy chain molecule and β2m was 30% and 25%,respectively,and their molecular weights were about 37KD and 12KD,respectively.The purity of sHLA-G1 heavy chain and β2m protein could also reach about 95%.Conclusion The prokaryotic expression vectors for heavy chain molecule and β2m of sHLA-G1 have been established successfully,providing firm basis for the clarification of the function of sHLA-G1.
【Fund】: 广东省自然科学基金项目(编号:05300928);; 华山医院院级课题(院193)
【CateGory Index】: R392
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【Co-references】
Chinese Journal Full-text Database 2 Hits
1 LI Zheng-yuan *,CHEN Gui-hua,LU Min-qiang. * Department of General Surgery Guangzhou Red Cross Hospital,Guangzhou 510080;Dynamic observation on the value of serum soluble human leukocyte class Ⅰ antigen after liver transplantation[J];Journal of Hepatopancreatobiliary Surgery;2004-01
2 IAN Jiang-Cai, WU Tao, ZHOU Hua-You , YANG Quan-Ke , ZHANG Yin-Ze, WU Da-Lin, WEI Ya-Ming, CAO-Qiong,XIA Rong. Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. Kelong Medical Apparatus Co. Ltd, Xinxiang 453400, China.;Detection for soluble HLA-I antigens and its dynamic monitoring of renal trans-plant rejection[J];Chinese Journal of Immunology;2004-01
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