Molecular Cloning And Sequence Analysis Of Musclin Of Different Animals

In order to study the function of the Musclin of pig, chicken, cattle, sheep and rabbit, according to mouse and rat Musclin gene sequence search the Musclin gene of pig, cattle, sheep, rabbits and chicken in their ESTs and UniGene .Using the searched sequences ,we design the primers.Then all the five animals'musclin gene was successfully cloned by RT-PCR. Finally choosing pig Musclin gene, we do prokaryotic expression, protein purification and identification by Western blotting. The results as follows:Molecular cloning and sequence analysis of sheep musclin.The results as follows: cloned sheep Musclin gene contains a complete open reading frame (open reading frame, ORF), the total length of 413 bp, encoding the 132 amino acid, including 19 acidic amino acids, 24 alkali amino acids. Its molecular weight is 14.53 kDa and isoelectric point is 10.926. The predicted signal peptide consists of 27 - 28 amino acids. Mature Musclin peptide contains 104 - 105 amino acid. The amino acid sequence contains "KKKR" structure and homology region with ANP, BNP, CNP of mouse by analysis.Molecular cloning and sequence analysis of rabbit musclin.The results as follows: cloned rabbit musclin gene contains a complete open reading frame. The length is 409 bp, encoding a 132 amino acids. Identity analysis show that the rabbit musclin nucleotide sequence share 78.2%, 79%, 87.9%, 98.2% and 46.7% homology with that mouse, rat, human, pig and chicken, respectively. Tthe deduced protein sequence share 73.1%, 74.2 %, 87.9%, 96.2% and 56.8% homology with that of mouse, rat,human, pig and chicken, respectively. The deduced peptide contains a"KKKR"motif and the region homologous to mouse ANP, BNP, and CNP.Molecular cloning and sequence analysis of chicken musclin.The results as follows: cloned chicken Musclin gene contains a complete ORF. The length of ORF is 402 bp, encoding a 133 amino acids. The predicted signal peptide is 1– 30aa. The deduced peptide contains a"KKKR"motif and the region homologous to mouse ANP, BNP, and CNP.Identity analysis show that the chicken musclin nucleotide sequence share 59.8%,61.2%,63.9%,62.2% and 61.3% homology with that mouse, rat, human, pig and rabbit, respectively. The deduced protein sequence share 58.6%,53%,51.5%,58.3% and 56.8% homology with that of mouse, rat,human, pig and rabbit, respectively.Molecular cloning and sequence analysis of cattle musclin. The results as follows: Cloned cattle Musclin gene contains a complete open reading frame (open reading frame, ORF), the total length of 413 bp, encoding the 132 amino acid . including 19 acidic amino acids, 24 alkaline amino acids. Its molecular weight is 14.53 kDa and isoelectric point is 10.926. The predicted signal peptide 27-28 amino acids. Mature Musclin protein contains 104 - 105 amino acid. The amino acid sequence contains "KKKR" structure and homology region with ANP, BNP, CNP of mouse by analysis. Identity analysis show that the deduced protein sequence share 93.2%, 80.3%, 78.5%, 84.1% and 60.6% homology with that human, rat, mouse, pig and chicken respectively. The predicted signal peptide share 93.3%, 78.8%, 76%, 84.6% and 67.6% homology with that of human, rat, mouse, pig and chicken, respectively. The mature Musclin protein share 93.3%, 78.8%,76%,84.6% and 67.6% homology with that human, rat, mouse, pig and chicken ,respectively.Molecular cloning and sequence analysis of pig musclin.The results as follows: Cloned pig Musclin gene contains 651 bp. The open reading frame analysis includes 399 bp (54-452 bp) ,encoding 132 amino acids. And human, mouse and rat skeletal muscle of cDNA encoding a higher homology of homology were 87.2%, 77.6% and 77.9%. Speculate pig muscle-length amino acid sequence of 132 aa, The predicted signal peptide is 1 to 27 aa by SignalP. The deduced peptide contains a"KKKR"motif and the region homologous to mouse ANP, BNP, and CNP. The deduced protein sequence share 87.1%, 72.3% and 72.7% homology with that of human ,mouse and rat, respectively.According to the sequence,we design primers,then use RT-PCR to identify. The result shows it is correct.Then the gene fragment was cloned into expression vector pGEX-4T-1,and the recombinant was transformed into E coli BL21.Induced by IPTG,the fusion protein GST-musclin,a 38.59 kDa protein was successfully expressed and purified, then identified by Western blotting.