Cheong H, Gill S S
Department of Entomology, University of California, Riverside 92521, USA.
Appl Environ Microbiol. 1997 Aug;63(8):3254-60. doi: 10.1128/aem.63.8.3254-3260.1997.
A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B. thuringiensis subsp. jegathasan was cloned employing a limited-growth PCR screening method with forward and reverse oligonucleotide primers designed from N-terminal amino acid sequences of native and trypsin-cleaved protein, respectively. The expressed protein showed little cross-reactivity to the antibody raised against the Cyt1Aa protein. Unlike Cyt1Aa and Cyt2Aa expression, there was little or no visible crystal inclusion formation under microscopic observation. The amino acid sequence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, respectively. The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions between alpha-helices and beta-sheets and one in the loop region between beta-sheets 5 and 6. beta-Blocks 4 to 7 are also conserved, not only structurally but also among the amino acids in the hydrophobic faces. Mosquitocidal activity assays indicated that the Cyt2Bb toxin had less toxicity than Cyt1Aa and had about 600-times-lower toxicity than the wild-type whole toxin crystal. However, both the Cyt2Bb and the Cyt1Aa toxin showed comparable levels of hemolytic activity.
利用有限生长PCR筛选方法，从苏云金芽孢杆菌jegathasan亚种中克隆出一个编码30.1 kDa Cyt2Bb1毒素蛋白的溶细胞毒素基因，正向和反向寡核苷酸引物分别根据天然蛋白和胰蛋白酶切割蛋白的N端氨基酸序列设计。表达的蛋白与针对Cyt1Aa蛋白产生的抗体几乎没有交叉反应。与Cyt1Aa和Cyt2Aa的表达不同，在显微镜观察下几乎没有可见的晶体包涵体形成。氨基酸序列比对表明，与Cyt1Aa和Cyt2Aa的同一性分别为31%和66%。对五种已知溶细胞蛋白的序列比对表明有三个高度保守区域，两个在α螺旋和β折叠之间的环区域，一个在β折叠5和6之间的环区域。β链4至7也保守，不仅在结构上，而且在疏水面上的氨基酸之间也保守。杀蚊活性测定表明，Cyt2Bb毒素的毒性低于Cyt1Aa，比野生型全毒素晶体的毒性低约600倍。然而，Cyt2Bb和Cyt1Aa毒素的溶血活性水平相当。
