贝氏隐孢子虫Rhomboid基因的克隆与生物信息学分析  

Molecular cloning and bioinformatics analysis of Rhomboid gene in Cryptosporidium baileyi

作　　者：薛雪[1] 阳毅敏[1] 陈学秋[1] 杜爱芳[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310058

出　　处：《中国兽医学报》2015年第2期235-241,共7页Chinese Journal of Veterinary Science

基　　金：浙江省重大科技专项重点农业资助项目(2012C12009-2)

摘　　要：将Gen Bank中发表的C.parvum、C.hominis和C.muris的菱形体蛋白(Rhomboid,ROM)基因序列进行比对分析,设计合成该基因保守片段的1对特异性引物,以贝氏隐孢子虫基因组DNA为模板,PCR扩增出CbROM基因保守片段,采用染色体步移技术获得基因片段3'端和5'端核苷酸序列,并对所得序列进行测序、拼接与生物信息学分析。结果显示,CbROM基因ORF长度为1 422 bp,编码473个氨基酸,蛋白质理论相对分子质量为52 700,等电点为8.65,是疏水性蛋白质,含有7个串连的跨膜螺旋区,无信号肽;该基因含有典型的菱形体蛋白相关保守结构域,属于菱形体蛋白酶家族;功能位点分析结果显示,CbROM蛋白序列包含6个N-糖基化位点,2个c AMP、c GMP依赖性蛋白激酶磷酸化位点,8个蛋白激酶C磷酸化位点,7个酪蛋白激酶II磷酸化位点和10个N-豆蔻酰化位点;二级结构中包含α-螺旋38.27%,β-折叠16.7%和无规则卷曲45.03%。本试验获得的贝氏隐孢子虫的Rhomboid基因序列,为该基因功能的进一步研究及其基因工程疫苗的研制奠定基础。Comparing the sequences of Rhomboid gene of Cryptosporidium parvum,Cryptosporidium hominis and Cryptosporidium muris in Gen Bank,we got the conserved fragment of the Rhomboid gene in Cryptosporidium baileyi. Then,3' and 5' flanking regions of the conservative sequences were amplified using the genome walking technique based on polymerase chain reaction( PCR) method. After overlapping and assembling,the complete sequence of CbROM gene was obtained. According to the bioinformatics analysis,CbROM contains a 1 422 bp open reading frame( ORF),which encodes a hydrophobic protein of 473 amino acids with a predicted molecular weight of 52 700 and an isoelectric point( p I) of 8. 65. The protein has 7 transmembrane helices and is absence of signal peptide cleavage sites. Besides,it also contains a conserved Rhomboid-like domain and is a member of Rhomboid protease family. Functional analysis showed that there are 6 N-glycosylation sites,2 c AMP- and c GMP-dependent protein kinase phosphorylation sites,8 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites and 10 N-myristoylation sites in CbROM. In addition,its secondary structure comprises 38. 27% α-helices,16. 7% β-sheets and 45. 03% irregular coils. We have reported the Rhomboid sequence of C. baileyi,and the results would lay the foundation for further research in the gene function and the further development of genetic engineering vaccines.

关 键 词：贝氏隐孢子虫 Rhomboid基因 染色体步移 生物信息学分析 

分 类 号：S852.723[农业科学—基础兽医学]