Cloning And Bioinformatic Analyses Of Preprochymosin Of Xinong Saanen Goat And Construction Of Eukaryotic Expression

The abomasums of 5 d postparturition Xinong Saanen Goat was sampled with biopsy. The total RNA was isolated with Trizol and the RNA quality was detected by gel electrophoresis. Then based on blast the sequence of Ovis aries preprochymosin cDNA (Accession No: X53037) reported in GeneBank, two pairs of primers were designed to clone Xinong Saanen Goat preprochymosin with the 5 d postparturition abomasum cDNA as template by RT-PCR method. The PCR product was ligated to T-vector and successfully cloned in E.coli. After sequencing, the goat preprochymosin nucleotide and protein sequence homology analysis were proceeded with NCBI BLASTn, BLASTp and ClustalW. The softwares Compute pI/MW, MLRC and SignalP V3.0 were used to analyze the physical property, secondary structure, folding type and signal peptide. The tertiary structure and active center site was predicted using FeatureMap3D and SWISS-MODE. Based on the sequencing result, the primer was designed, and the yeast expression vector pPICZαA was to construct the recombinant plasmid. The recombinant plasmid was integrated into Pichia pastoris DNA using electro electro-transformation and the positive recombinants of preprochymosin were screened and identified. The results showed that:1. The results indicated that the goat preprochymosin cDNA had 1292 nt which coded 381 amino acid residues and it has been registered in Genbank with accession number EF199763. The nucleotide alignment showed that, the homologies nucleotide and peptide sequence of the Xinong Saanen Goat preprochymosi with the other the goat, bovine, sheep, bubal, camel, dog, human, pig, rhesus and rabbit were 99.41%,98.74%,95.29%,95.81%,87.48%,83.69%,81.91%,67.43%,66.74%,66.39% and 99.21%,98.42%,93.70%,93.42%,83.99%,74.09%,57.22%,56.37%,57.18%,54.18%.2. The results of physical property analysis indicated that of preprochymosin of Xinong Saanen Goat were 42.1kDa. The molecular weights of chymosin were 35.5kDa and its isoelectric point was 4.45.3. The second structure and folding type analysis results of preprochymosin showed, 8.36% of total amino acid wasαhelix and 32.82% wasβsheet. Inαhelix, there was not same fragment between the N end of 1-175 amino acid and the C end of 176-323.4. The results of signal peptide prediction indicated that there was a signal peptidethe which was consisted of 16 amino acids and cleavage site was located between Met16-Gly17.5. The 224th amino acid in the mature chymosin formed from the cloned preprochymosin was Gly and the results of tertiary structure predictiones showed that the chymosin would form B typr chymosin. Two aspartate activity sites Asp32, Asp215 and the amino acid Thr33, Ser35, Thr216, Thr218, NHGly217, NHGly34 which maintain the three dimension conformation of active center, and disulfide bond sites Cys45-Cys50, Cys206-Cys210, Cys250-Cys283 were matched with the predecessor's researchs, This revealed the chymosin could form the right three dimension conformation with bilobular structure.6. The non fusion and fusion protein recombinant expression vectors pPICZ-T1 and pPICZ-T2 of goat preprochymosin were successfully constructed using yeast secreted expression vector pPICZαA. After the electro-transformation, the preprochymosin recombinant expression plasmid pPICZ-T1and pPICZ-T2 were integrated into the genome of Pichia Pastoris and the non fusion and fusion protein recombinants which could expressed the goat preprochymosin were screened out. And this will supply the experimental data for chymosin expression research and industrialized production.