Cloning And Prokaryotic Expression Of The CRD Of Chinese Marmota Asialoglycoprotein Receptor H1 And H2 Subunit, And Preparation And Identification Of Polyclonal Antibody

Objective To construct the prokaryotic plasmids expressing the carbohydrate recognition domain(CRD) genes of the Chinese marmota asialoglycoprotein receptor(ASGPR) H1 and H2 subunit,express and purify the purpose protein ASGPR CRDH1 and CRDH2, immunize BALB/C mice, generate polyclonal antibodies against ASGPR CRDH1 and CRDH2, detect the PAbs.Methods ASGPR CRDH1 and CRDH2 gene were amplified by reverse transcriptase polymerase chain reaction, from the liver of Chinese marmota .Then in vitro the purpose gene were cloned into MCS region of prokaryotic vector—pRSET-B respectively. Induced by IPTG, the purpose protein were expressed in E.coli strain BL21(DE3)plysS, respectively in abundance. The antigenicity of the purpose protein purified to a high purity by Ni²⁺-chromatography was analyzed by Western blot. Polyclonal antibodies was developed by immunizing BALB/c mice with purified and denatured recombinant protein, the sensitivity and specificity were tested by ELISA, IHC and Western blotting analysis.Results 1. The pRSET-B.CRDH1 and pRSET-B.CRDH2 prokaryotic plasmid was successfully constructed and confirmed by RT-PCR, endonuclease digestion and direct sequencing.2. Being analyzed by SDS-PAGE and Western blot, The purpose protein could express in abundance in the form of inclusion bodies. We got the purified purpose protein by Ni²⁺- chromatography.3. Ponoclonal antobodies against the carbohydrate recognition domain(CRD) genes of the Chinese marmota asialoglycoprotein receptor(ASGPR) H1 and H2 subunit were established. The PAbs had a high specificity and high titerl, which was indentified by ELISA and IHA.Conclusions1. The expression vectors of recombinant pRSET-B.CRDH1 and pRSET-B.CRDH2 were constructed successfully.2 .Immunized with the purpose protein exprsessed, BALB/C mice generate two polyoclonal antibodies against the carbohydrate recognition domain(CRD) genes of the Chinese marmota asialoglycoprotein receptor(ASGPR) H1 and H2 subunit respectively. The pAbs have a high specificity and high titer .SignificanceRecombinant ASGPR CRDH1 and CRDH2 protein expressed in this study has a potential value as a targeting molecule for gene therapy and research of liver diseases。Immunized with the purpose protein expressed, BALB/C mice generated two polyclonal antibodies against ASGPR CRDH1 and CRDH2 protein .The results of our experiment provid persuasional evidence for the application of these polyclonal antibodies in the future.