滨藜（Atriplex patens）核糖体失活蛋白基因的克隆和分析

为开发利用新疆野生植物滨藜，采用反转录-多聚酶链式反应(RT-PCR)，从滨藜（Atriplex patens）叶中克隆核糖体失活蛋白（ribosome inactivating proteins ，RIPs）基因的完整读码框ORF片段.序列分析表明，该片段大小为843 bp,编码1 条长280个氨基酸肽链, 其中N 端的26个氨基酸是信号肽.滨藜核糖体失活蛋白（ApRIP）属Ⅰ型核糖体失活蛋白.同源性比较分析显示，滨藜RIP与藜科植物藜的核糖体失活蛋白，天花粉蛋白（TCS）和美洲商陆蛋白（PAP）基因序列的同源性分别为82%，41%和55%.氨基酸序列比对和SWISS-MODEL同源模建分析表明，滨藜RIP的3′端氨基酸肽链具有与其它RIP相同的活性中心位点"EAARXKXI".

Cloning,Expression of Ribosome Inactivating Proteins Gene from Atriplex patens in Xinjiang

The complete open reading frame (ORF) for the ribosome-inactivating proteins (RIPs) in the Atriplex patens leaves was obtained by RT—PCR．The sequence data showed that the cloned fragment is 843bp. The open reading frame of the fragment contains 281 amino acid residues and there is a signal sequence with 26 amino acid residues at its N-terminus. The analysis suggests that ApRIP belongs to type ⅠRIP. The amplified fragment has 82%，55% and 41% identity comparing with RIP gene nucleotide of Chenopodium album, pokeweed antiviral protein and Trichosanthes kirilowii trichosanthin, respectively.The amino acid sequence and SWISS-MODEL analysis indicated that the “EAARXKXY”is active centre of the enzyme in RIPs. The IPTG inducible expression vector containing the RIP gene was constructed and transferred into E coli BL21(DE3).The specific protein was produced after inducing with 0．4mmol／L IPTG SDS—PAGE of the fusion protein demonstrated that the RIP migrated at a size of 77kDa．The immunization was performed by intra—muscular injection of pCDNA3-Aprip，and then antiserum was detected by ELISA．Western blotting analysis showed the antiserum was specific against the ribosome inactivating proteins．