H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2018.11.002

Abstract

Abstract:

This experiment was aimed to express recombinant neuraminidase(NA) protein of H6N6 subtype avian influenza virus (AIV) isolated from Guizhou province,and prepared polyclonal antibody against NA protein of AIV.The specific primers were designed according to N6 gene of AIV in GenBank(accession No.:MG434500),and used for amplification of N6 gene by PCR.PCR product was cloned into the expression vector pET-32a(+).The soluble recombinant protein was expressed in E.coli BL21(DE3) with IPTG.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein.The titer of the antibody was detected by indirect ELISA.The results showed that coding region of N6 gene was 1 380 bp,which encoded 459 amino acids.There were 33 nucleotides deletion from position 175 to 207 bp at N6 gene stalk.The recombinant plasmid pET-32a-N6 was identified by double enzyme digestion,it appeared about 5 900 bp vector band and about 1 380 bp target band,pET-32a-N6 recombinant plasmid was successfully constructed;SDS-PAGE analysis result showed the molecular weight of the recombinant N6 protein was about 70 ku which consistented with expected result.It showed that the target gene was successfully expressed in the form of inclusion body.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 70 ku in size,indicating that the purified protein was the recombinant protein pET-32a-N6.The purified protein of denaturation and renaturation could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum,respectively.The titer of the antibody was about 1:3 200 by detection of indirect ELISA.All the results indicated that N6 gene of H6N6 subtype AIV was successfully cloned and expressed,the prepared anti-N6 showed good immune activity,and the protein purified could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum.

Key words: avian influenza virus (AIV); N6 subtype; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺7

WAN Run, HUA Min, LONG Lishu, HE Xinwei, LUO Yinxing, ZHOU Bijun, WANG Kaigong, CHENG Zhentao, WEN Ming. Prokayotic Expression and Polyclonal Antibody Preparation of N6 Gene of H6N6 Subtype Avian Influenza Virus[J]. , 2018, 45(11): 2989-2995.

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Prokayotic Expression and Polyclonal Antibody Preparation of N6 Gene of H6N6 Subtype Avian Influenza Virus

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