微小牛蜱Bm91基因的克隆及其真核表达载体的构建

Cloning of Bm91 gene of Boophilus microplus and construction of eukaryotic expression vector with Bm91 gene

参照GenBank中收录的微小牛蜱Bm91基因的核苷酸序列,设计了1对特异性引物,从我国微小牛蜱半饱血雌性成蜱肠道和唾液腺研磨物中提取总RNA,利用M-MLV RTase cDNA Synthesis Kit合成双链cDNA,并采用PCR技术扩增获得的Bm91基因,将其克隆到pGEM-T Easy载体中并进行PCR、酶切和测序分析鉴定.结果表明:所克隆的Bm91基因序列与GenBank上收录的Bm91基因序列的同源性达97.1%,而且该片段含有完整的开放阅读框;将该基因从克隆载体上用EcoRⅠ单酶切消化并亚克隆到巴斯德毕赤酵母分泌型表达载体pP1C9K的EcoRⅠ酶切位点,再次进行PCR、酶切和测序分析鉴定,结果表明成功构建并获得了微小牛蜱Bm91基因的重组真核表达载体pP1C9K-Bm91.

The total RNA was extracted from the salivary glands and the intestine of semiengorged adult female Boophilus microplus and double eDNA was synthesized using M-MLV RTase eDNA Synthesis Kit. According to the nucleotide sequences of Bin91 published in GenBank, a pair of primers was designed and a 1 908 bp Bin91 gene was amplified by PCR. The target gene was subcloned into pGEM-T Easy vector and tested by PCR, enzyme digestion and sequencing. The sequencing showed that the cloned Bin91 gene shared 97.1 % homology with the date published in GenBank and this fragment contained the complete open reading frame of Bin91 gene. Then the gene was cleaved from pGEM-T Easy vector by EcoR I and subcloned into pPIC9K secretory expression vector of Pichia pastoris. The recombinant eukaryotic expression vector of designated pPIC9K-Bmgl was constructed successfully.