Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota himalayan.
The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.
Yang
Yan
Y
Center of Experimental Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. yyang@tjh.tjmu.edu.cn
Huang
Huang
H
Zhang
Zhenghua
Z
Wang
Baoju
B
Tian
Yongjun
Y
Lu
Mengji
M
Yang
Dongliang
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D037263:Asialoglycoprotein Receptor / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006505:Hepatitis / Q000628:therapy; D008099:Liver / Q000378:metabolism; D008392:Marmota / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology