抗对虾白斑综合症病毒单链抗体A1基因在酵母中表达及其与抗原结合活性
EXPRESSION OF ScFv-A1 OF AGAINST-WHITE SPOT SYNDROME VIRUS(WSSV)IN YEAST PICHIA PASTORIS
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摘要: 通过PCR从噬菌粒载体上扩增一种抗对虾白斑综合症病毒(WSSV)的单链抗体A1(ScFvA1)基因,并构建于大肠杆菌-酵母穿梭质粒载体pPIC9K上.经PCR,酶切,测序鉴定重组克隆,发现重组成功.将重组质粒pPIC9K-ScFvA1转化毕赤酵母(Pichia pastoris)GS115中,利用甲醇诱导,将单链抗体A1在酵母中进行了初步表达.经SDS-PAGE电泳,发现其大小约为32KD,通过ELISA实验,证明表达上清液中的单链抗体具有很高的WSSV结合活性.
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关键词:
- 毕赤酵母:对虾白斑综合症病毒(WSSV) /
- 单链抗体 /
- 克隆 /
- 表达
Abstract: White spot syndrome virus(WSSV)from shrimp was first found in Taiwan in 1992.Since 1993,white spot syndrome disease of shrimp caused by this virus was widely spread throughout China,Asia and Pan-Pacific Ocean It is a great harm to the aquaculture of shrimp.Unfortunately,we still haven’t found a powerful method to protect or treat shrimp from this disease.So it is important to detect this virus in short time.In our research,a gene of ScFv-A1 against White spot syndrome virus(WSSV)from shrimp was amplified by PCR from phagemid vector pCANTAB5E and cloned into the E.Coli-yeast shuttle vector-pPIC9K,yielding expression vector pPIC9K-ScFv-Etag.The correct inserting was confirmed by PCR,BglⅡ enzyme digestion and DNA sequencing.Then the recombinant vector pPIC9K-ScFv-Etag was transformed into the yeast strain GS115(Pichia pastoris GS115)by sephroplasting and the ScFvA1 was expressed under the control of the AOX1 promoter.After induced by 2%methanol every 24 hours for 96 hours,the ScFvA1 could be secreted into the supernatant.From the SDS-PAGE we found that the molecular weight of the protein was about 32KD.Its WSSV binding activity could be detected by ELISA.And we found that its WSSV binding was 6—7 times higher than negative comparison.The result suggests that the genetically engineered single chain antibody can be expressed in the eucaryotic system successfully.It means that we can use yeast as a expression system to produce the antibody to detect,prevent,and inhibit the White spot syndrome virus in the near future.-
Keywords:
- Pichia pastoris /
- White Spot Syndrome Virus(WSSV) /
- Single-chain Fv(ScFv) /
- Cloning
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