几株杆状病毒包涵体蛋白双向高压电泳指纹图谱比较

Comparative Study of the Fingerprinting Map of Granulins and Polyhedrins

用蔗糖密度梯度离心提纯的6种昆虫病毒包涵体(BsNPV1,BsNPV2,BtNPV,EpNPV,PrGV,PxGV)经DAS碱解,粗提纯的包涵体蛋白经Sepharose-6B柱层析和Caaalco-prep-disc法进一步纯化后,在SDS-PAGE中显示为一条分子量约28,000d的带。用常规双向免疫扩散法检查各种包涵体蛋白之间的差异,结果表明血清学BsNPV与BtNPV,PrGV与PxGV之间都是不可分辩的。6种包涵体蛋白的双向高压指纹图谱表明,它们的指纹图谱都不同程度地存在着一些相同或十分相似的肽点。就每种病毒的指纹图谱又都是独特的,NPV间相似性低于GV间相似性。我们认为NPV和GV的包涵体蛋白在结构上存在着一些相同的或相似的保守区域,但不同种之间在整个一级结构上是有差异的。利用包涵体蛋白的指纹图谱鉴定杆状病毒是个灵敏可行的方法。

Six kinds of baculoviruses(BsNPV1, BsNPV2, BtNPV, EpNPV, PrGV, PxGV) are purified by centrifugation using sucrose gradient,Roughly purified granulins and polyhedrins are obtained by having the inclusion body solubilized in dilute alkaline salne after possible alkaline protease activity is inhibited. Then granulins and polyhedrins in highly pure form are obtined by using Sepharose-6B chromatography and SDS-polyacrylamide prep-disc(Canalco). Each kind of granulins and polyhedrins shows only one band on SDS-PAGE using the procedures of Laemmli.And the granulins and polyhedrins exhibit an estimated MW of 28,000.Two dimensional high-voltage electrophoresis(HVE) of highly purified granulins and polyhedrins preparations reveals relateness as well as differences among the proteins assessed by electrophoretic migration patterns.Double diffusion analysis shows cross-reaction between different granulins and polyhedrins.For example, PxGV can not be distingushed from PrGV and neither can BsNPV be distingushed from BtNPV.But using HVE technique, all of them can be distingushed from each other easily. According to these results, we suggest that the HVE technique be an excellent method in the classification and identification of baculoviruses.