Dayaoshan Asian Trees Shrew TLR3 Gene Cloning And Prokaryotic Expression

Objective:To clone Asia Guangxi Yao mountain shrew trees Toll-like receptor 3 (Toll like receptor 3, TLR3) open reading frame gene (Open Reading Frame, ORF) sequence, bioinformatic analysis. Construction of Guangxi Dayaoshan Asia trees shrew TLR3 gene ORF prokaryotic expression vectors, prokaryotic expression trees Asia Guangxi Yao mountain shrew Toll-like receptor 3 recombinant protein.Method:(1) Referring to Yunnan Valencia kinds TLR3 mRNA sequence prediction by Vector NTI analyze the ORF, and apply the primer design software Primer Premier 5.0 for the tree shrews TLR3 gene primers were designed, and the trees Asia Guangxi Yao mountain shrew peripheral blood first strand synthesis RNA template cDNA. (2) Design of nine specific primers amplified fragment, and fusion expression vectors PBET-4 connection. The recombinant plasmid PBET-4-TLR3 ORF into E. coli DH5a, LB medium containing Amp sieve out monoclonal colonies colony PCR identification of recombinant clones molecular size. (3) Get sequencing, gene by comparative analysis of Guangxi Dayao Mountain sub trees shrew TLR3 gene ORF sequences and predict Yunnan Valencia trees shrew TLR3 gene ORF sequence homology, and Guangxi Dayaoshan Asian shrew TLR3 gene trees Bioinformatics Analysis. (4) the recombinant plasmids PBET-4-TLR3 ORF, will be transferred into the expression strain DE3, induced by IPTG. After protein denaturation was prepared for protein SDS-PAGE gel electrophoresis, and then stained with Coomassie Brilliant Blue bleaching analysis Dayaoshan Asia Guangxi trees shrew Toll-like receptor 3 recombinant protein expression.(5) the use of software DNAMAN Asia Guangxi Yao mountain trees shrew TLR3 gene ORF sequences cloned gene sequence analysis; using the NCBI BLAST tool on the website of Guangxi Yao mountain trees Asian shrew TLR3 gene ORF sequences with other species homology analysis. Using Vector NTI software for the amino acid sequence of Guangxi Dayao Mountain Shrew TLR3 protein sub-trees to predict, and the Guangxi and Yunnan Dayaoshan West Asia planting trees shrew shrews and amino acid sequences were compared TLR3 gene ORF; by software MEGA 5.10 phylogenetic tree; use (http://web.expasy.org/protscale/) online software forecast Asia Guangxi Yao mountain trees shrew TLR3 protein hydrophobic; use (http://smart.embl-heidelberg.de/) online software forecast Asia Guangxi Yao mountain trees shrew TLR3 protein critical domains; with online analysis tools NetPhos 2.0 Server for phosphorylation sites of Guangxi Dayao Mountain shrew TLR3 protein sub-trees to predict.Results:(1) by sequencing and comprehensive analysis showed that the prokaryotic expression vector PBET-4-TLR3 ORF plasmid properly connected and trees Asia Guangxi Dayao Mountain Shrew TLR3 gene ORF sequences of full length 2757bp, A+T content of 59.15%, C+G content of 40.85%. Encoding 918 amino acids; after expression strain induced by IPTG (2) turn have PBET-4-TLR3 ORF of DE3, showed trees Asia Guangxi Dayao Mountain Shrew TLR3 recombinant protein successfully expressed molecular size of about 125kDa, with expectations theoretical value in line with the target protein. Guangxi Dayaoshan Asian trees Shrew TLR3 protein analysis showed: molecular 105.3041kDa, isoelectric point of 8.31; hydrophobic scores between-3.0 to 4.0; the transmembrane protein structure, and there are 19 key domains wherein the intracellular segment of the TIR, extracellular domain and intracellular domain, respectively,18 and a key domains; it has also found that 37 phosphorylation sites, which contains three threonine protein kinase phosphorylation site points, eight tyrosine protein kinase phosphorylation sites and 27 serine protein kinase phosphorylation sites. (3) sub-trees Guangxi Yao mountain shrew TLR3 gene ORF sequences bioinformatics analysis such as: gene sequence alignment showed that Guangxi and Yunnan Dayao Mountain West Asia planting trees shrew shrew TLR3 gene sequence similarity highest 98.33%, but there are still differences; and humans, chimpanzees, monkeys, dogs, pigs similarity followed for 84.75%,84.71%,84.50%,84.48%,84.84%, and the lowest similarity rodents. By amino acid sequence homology than was found in Guangxi and Yunnan Dayaoshan subspecies West shrew TLR3 trees belong to the same groups, and monkeys, chimpanzees and people from the past, confirmed Guangxi Dayaoshan Asian shrew trees close to primates animal;Conclusion and Significance:the first successful cloning of Guangxi Dayao Mountain sub trees shrew TLR3 gene ORF sequences and prokaryotic expression vector PBET-4-TLR3 ORF, obtained expression induced E. coli BL21 (DE3) at a molecular weight of about the size of about 125kDa specific protein bands. Guangxi and Yunnan Dayaoshan West Asia planting trees shrews shrew TLR3 protein 11 different amino acids, and therefore both TLR3 may differ antigenically. FQ primer design, polyclonal and monoclonal and functional studies of this study for subsequent sub-trees in Guangxi Yao mountain shrew TLR3 antibody gene basis.