Construction of a cDNA library from Agkistrodon acutus venom gland and identification of Agkihagin, a novel transcript for metalloproteinase
- VernacularTitle:尖吻蝮毒腺cDNA文库的构建、金属蛋白酶基因克隆和序列分析
- Author:
Qinghua LIU
;
Songnian HU
;
Wei YIN
;
Xingwen SU
;
Xiaowei ZHANG
;
Chenji LI
;
Pengxin QIU
;
Guangmei YAN
- Publication Type:Journal Article
- Keywords:
gene library;
Agkistrodon acutus;
me-talloendopeptidase;
sequence analysis
- From:
Chinese Journal of Pharmacology and Toxicology
2006;20(2):81-90
- CountryChina
- Language:Chinese
-
Abstract:
AIM To construct a non-normalized cDNA library from Agkistrodon acutus venom gland as an imtial step to develop new and more effective venom by genetic engineering technique for screening and expressing target genes. METHODS The total RNA was extracted from fresh venom gland using Trizol. mRNA was reversely transcripted to cDNA using superscriptⅡ reverse transcriptase. Second-strand synthesis was performed using DNA polymeraseⅠ. After adding EcoRⅠ adaptor, phosphorylating the end and digesting with XhoⅠ, the cDNA was collected in five fractions (<0.25 kb, 0.25-0.5 kb, 0.5-1 kb, 1-2 kb and >2 kb) using the QIAquick Gel Extraction kit and ligated to pBluescriptⅡ vectors. The five libraries obtained were plated by infecting E.coli DH10B, constructing a cDNA library of Agkistrodon acutus venom gland. Sequencing clones at random, 8696 high quality 5′ end expressed sequenced tags (ESTs) were obtained and analyzed. The initial sequences were assembled into 2855 clusters. Among which, one of the clusters (Agkihagin) consisting of 74 ESTs was identified as a novel metalloprtoteinase based on RT-PCR and sequence analysis. RESULTSThe titers of library were 2.048×106. The novel metalloproteinase belonged to PⅢ type metalloproteinase. Its open reading frame was composed of 1827 nucleotides and coded a pre-zymogen of 608 amino acid with zinc-binding domain for metalloproteinase and Asp-Glu-Cys-Asp(DECD) domain for disintegrin. CONCLUSION The capacity of cDNA library of venom gland is above the general level of cDNA library. It would be a helpful platform to construct a catalog for transcripts in the venom gland of the Agkistrodon acutus. The sequence analysis indicates that the deduced amino acid sequence of the identified gene for metalloproteinase share the highest 87% identity with the metalloproteinase genes of other snakes in the GenBank. It lays a good foundation for the study of structure-function relationships of snake venom metalloproteinases.
