Cloning and expressing of tissue inhibitor of metalloproteinases Ⅰ gene fragment and preparation of monoclonal antibodies against the recombinant protein
10.3760/cma.j.issn.1003-9279.2013.03.024
- VernacularTitle:人基质金属蛋白酶抑制剂Ⅰ原核核表达系统的构建及单克隆抗体的制备
- Keywords:
Interstitial collagenase;
Cloning,molekular;
Antibody,monoclonal
- From:
Chinese Journal of Experimental and Clinical Virology
2013;27(3):231-233
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases Ⅰ (TIMP-Ⅰ)fusion protein.Methods TⅠMP-Ⅰ gene was amplified from fibrotic human liver tissue by RT-PCR,then ligated with pQE31 to form recombinant plasmid pQE-TIMP-Ⅰ and transformed into E.coli BL21.The protein induced by IPTG was purified by 6 × His-tag and used to immunize the BALB/c mice.The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique.Western Blot were used to detect specificity of mAbs.Results The prokaryotic plasmid expressing the recombinant protein was constructed,and the TIMP-Ⅰ recombinant protein was expressed and purified.Four hybridoma cell lines that secreted anti-TIMP-Ⅰ mAbs were obtained.3 of 4 mAbs were the IgG1 subtype.Western Blot indicated the mAbs showed specific combination with TIMP-Ⅰ protein.Conclusion The TIMP-Ⅰ recombinant protein is highly purified and has strong antigenicity.The anti-TIMP-Ⅰ mAbs were prepared successfully.
