野桑蚕卵黄原蛋白的鉴定及cDNA序列分析

利用SDS-PAGE和Western blot方法分析鉴定了野桑蚕Bombyx mandarina Moore卵黄原蛋白，发现该蛋白由大小两个亚基组成，分子量分别为175 kD和42 kD。利用昆虫卵黄原蛋白进化上的保守性，根据家蚕的卵黄原蛋白cDNA序列设计特异性引物在野桑蚕的总RNA进行RT-PCR扩增，对于3′和5′端进行RACE扩增，解析获得了野桑蚕卵黄原蛋白cDNA全序列(GenBank登录号AY 309967)。该序列含有5.754个碱基，由一个开放阅读框组成，编码1.780个氨基酸，卵黄原蛋白的氨基酸序列与家蚕的同源性达到97.6%。在特定的酶切位点(RSRR)处，即第364～367个氨基酸位置，卵黄原蛋白前体被酶切为大小两个亚基，根据氨基酸推算的相对分子质量分别为161.571 kD和40.794 kD，如果考虑到翻译后的修饰，这与SDS-PAGE的结果是吻合的。同源性分析表明，昆虫卵黄原蛋白一级结构分化基本上局限在同一目内，具有较高的保守性。

Identification and cDNA sequencing of vitellogenin in Bombyx mandarina Moore

The large subunit and small subunit of vitellogenin in Bombyx mandarina were identified through SDS-PAGE and Western blot, with molecular weight about 175 kD and 42 kD respectively based on high molecular weight marker. Several primers were designed according to cDNA sequence of vitellogenin in Bombyx mori and conservatism of insect vitellogenin. The cDNA sequence of vitellogenin in Bombyx mandarina (GenBank accession number: AY309967) was obtained through RT-PCR of the total RNA and RACE amplification of 3′end and 5′end. This sequence contains 5 754 bases, an open reading frame which encodes 1 780 amino acids, and the putative cleavage sites RSRR for the proteases of the subtilisin family at position 364－367 where provitellogenin is cleaved into two subunits. The molecular weights of the two subunits are 161.571 kD and 40.794 kD respectively based on amino acids, which are in accordance with the SDS-PAGE results if modification after translation is considered. The results of homology analysis suggested that amino acid sequence of insect vitellogenin be highly conserved within the same order for insects.