目前在昆蟲桿狀病毒表達系統(tǒng)（baculovirus expression vector system，BEVS）中表達A型塞內(nèi)卡病毒（Senecavirus A，SVA）結(jié)構(gòu)蛋白VP1尚未見報道。本研究將VP1基因通過限制性酶切位點插入桿狀病毒載體pFastBac I，然后將鑒定正確的重組載體轉(zhuǎn)化至DH10Bac感受態(tài)細胞，經(jīng)藍白斑篩選，對鑒定正確的菌落提取質(zhì)粒。將質(zhì)粒轉(zhuǎn)染Sf9昆蟲細胞，待有明顯細胞病變后收獲重組桿狀病毒rBac-I-VP1，并進行病毒滴度測定。隨后通過蛋白免疫印記（Western Blot）和間接免疫熒光（IFA）鑒定VP1蛋白表達情況。結(jié)果顯示，重組桿狀病毒rBac-I-VP1滴度為6.8×105 pfu/mL；Western Blot可檢測到相對分子質(zhì)量約27 kDa的表達產(chǎn)物，且其能夠被SVA兔陽性多克隆血清識別；IFA試驗顯示，VP1蛋白能夠在Sf9細胞內(nèi)表達。結(jié)果表明，本研究利用BEVS表達的SVA VP1蛋白具有良好的免疫反應(yīng)性，為其后期功能研究及SVA診斷試劑研制提供了技術(shù)基礎(chǔ)。
Expression and Identification of Senecavirus A Structural Protein VP1 in Insect Baculovirus
The expression of senecavirus A（SVA）structural protein VP1 in baculovirus expression vector system（BEVS）has been not reported till now. In the study，VP1 gene was inserted into baculovirus vector of pFastBac I through the restriction enzyme site，the identified recombinant vector was transformed into DH10Bac，and the correct colonies were screened by blue and white spots to extract the plasmids that were transfected into Sf9 insect cells，and the recombinant baculovirus rBac-I-VP1 was obtained after obvious cytopathic changes. The expression of VP1 protein was identified by Western Blot and indirect immunofluorescence（IFA）. The results showed that the titer of recombinant baculovirus rBac-I-VP1 was 6.8×105 pfu/mL；the expression products with relative molecular weight of about 27 kDa could be detected by Western Blot，which could be recognized by SVA rabbit positive serum；it was shown by IFA that，VP1 protein could be expressed in Sf9 cells. It was concluded that the SVA VP1 protein expressed by the BEVS was with good immunoreactivity，which provided a basis for future study on related functions and the development of diagnostic reagents.
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