鸡源SUMO标签蛋白的鉴定及在IBDV VP2原核表达中的促溶功能

Identification and functional analysis of SUMO-tag protein from chicken in improving expression level of soluble IBDV VP2 protein

VP2蛋白是传染性法氏囊病病毒(infectious bursal disease virus,IBDV)的保护性抗原。原核表达中能否获得具有天然构象的可溶性VP2蛋白是研发IBD亚单位疫苗的关键问题。为了解是否存在鸡自体促溶标签蛋白以及外源促溶标签能否在一定程度上促进蛋白的正确折叠,试验对不同物种来源的类泛素蛋白修饰分子(small ubiquitin-like modifier,SUMO)进行比对研究。结果显示,获取到的3种鸡源SUMO蛋白与酵母源SUMO蛋白的氨基酸同源性均高于40%,其中SUMO-1与SUMO-3两种蛋白可以在大肠杆菌中可溶性表达;通过SUMO-1、SUMO-3与VP2蛋白的融合表达,证明了鸡源SUMO蛋白对VP2具有促溶效果,得到的重组菌株P-28A-s1-VP2和P-28A-Os3-VP2琼脂扩散效价均达到1∶16;经过纯化后,融合蛋白SUMO-1-VP2的琼脂扩散效价可达到1∶256;使用SUMO蛋白酶酶切融合蛋白,SDS-PAGE和Western blot结果表明鸡源SUMO蛋白可以被成功切割,说明试验发现的鸡源SUMO蛋白可以作为促溶标签,在亚单位疫苗关键抗原表达中具有广阔的应用前景。

VP2 is identified as the major host-protective antigen of IBDV.Obtaining the soluble VP2 protein with natural conformation by using prokaryotic expression system is the key problem of the development of IBD subunit vaccine.To understand whether there is chicken derived tags and the exogenous tags can improve proteins folding correctly,small ubiquitin-like modifier(SUMO) derived from different species were comparatively analyzed in this study.The results showed that the amino acid homology of three chicken derived SUMO proteins acquired from NCBI was above 40%,compared to yeast derived SUMO,separately.The soluble SUMO-1 and SUMO-3 were expressed and obtained in Escherichia coli,respectively.The fusion of expression of SUMO-1/SUMO-3 with VP2 suggested that chicken derived SUMO protein can enhance the expression level of VP2.The titer of VP2 in both recombinant strains,P-28 A-s1-VP2 and P-28 A-Os3-VP2,can be induced to 1∶16 simultaneously by using agar diffusion method.After purified,the titer of the fusion protein SUMO-1-VP2 can reached up to 1∶256.The results of SDS-PAGE and Western blot also showed that SUMO-1-VP2 can be cleaved successfully by SUMO protease,proving that chicken derived SUMO protein can be used as soluble tags for future work.