[Soluble expression of A/H1N1 influenza virus HA with Drosophila S2 cell line and its bio-activity identification].

中华实验和临床病毒学杂志 Pub Date : 2013-10-01

Si-Min Yao, Qiao Lin, Guo-Liang Zhang, Hui Yang, Xiao-Feng Deng, Guang Nie, Xue-Bao Zheng, Ying-Xia Liu

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引用次数: 0

Abstract

Objective: To express soluble HA of A/H1N1 influenza virus in drosophila S2 cell line and identify its bio-activity.

Methods: HA gene was amplified from A/Shenzhen/71/09 virus strain using RT-PCR, then we constructed pAC5.1-HA expression vector, which was co-transfected into S2 cell with pCoblast vector. After transfection, stable S2 cell was selected through Blasticindin. HA in the supernatant was identified with Western Blot assay and purified with Ni-column. Recombinant HA was immunized into BALB/c mice 3 times, and the Abs titers were evaluated with ELISA.

Results: We successfully cloned HA gene with 1.7 x 10(3) bp of A/Shenzhen/71/09 virus strain and got recombinant pAC5. 1-HA expression vector. Stable S2 cell line was established after transfection and selection, which continuously expressed HA with molecular weight 75 x 10(3) D. After immunization with HA, the Abs titers were 1:1280 and 1: 5120 respectively on 10 d, 30 d.

Conclusion: We expressed soluble HA with good bio-activity, which contributed to research on immune diagnosis, subunit vaccine, and monoclonal Abs for influenza.

本刊更多论文

A/H1N1流感病毒HA在果蝇S2细胞株的可溶性表达及其生物活性鉴定

目的:在果蝇S2细胞系中表达甲型H1N1流感病毒可溶性透明质酸并鉴定其生物活性。方法:采用RT-PCR方法从A/Shenzhen/71/09病毒株中扩增HA基因，构建pAC5.1-HA表达载体，与pCoblast载体共转染S2细胞。转染后，通过Blasticindin筛选稳定的S2细胞。Western Blot法鉴定上清中的HA，镍柱纯化。重组HA免疫BALB/c小鼠3次，ELISA检测其抗体滴度。结果:成功克隆了A/Shenzhen/71/09病毒株1.7 × 10(3) bp的HA基因，获得重组pAC5。1-HA表达载体。经转染和筛选，建立了稳定的S2细胞系，连续表达分子量为75 × 10(3) d的HA。经HA免疫后，10 d、30 d的抗体滴度分别为1:1280和1:5 120。结论:表达的可溶性HA具有良好的生物活性，可用于流感的免疫诊断、亚单位疫苗和单克隆抗体的研究。

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来源期刊

中华实验和临床病毒学杂志

CiteScore

0.20

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0.00%

发文量

4549