Cloning, Recombinant Expression And Functional Analysis Of Genes Related To Immunity Of Chinese Shrimp, Fenneropenaeus Chinensis

Based on the techniques of molecular cloning and recombination, several genes related to immunity were cloned from Chinese shrimp (F. chinensis) such as antimicrobial protein (CruFc-1) and FcFer. The mature peptides of CruFc-1, CshFc, FcFer and MIH were subcloned into the prokaryotic expression vector and then transformed into host E. coli to express the recombinant protein, respectively. The mature peptide of CruFc-1 was subcloned into the eukaryotic expression vector and transformed into the host of Pichia pastoris KM71 to express the recombinant protein and then the recombinant protein was successfully obtained in 5 L fermenter. The promoter of CruFc-1 was obtained based on the technique of genome walking. The main results are introduced as follows: CruFc-1 and CshFc were cloned and characterized from the hemocytes cDNA of F. chinensis. The full-length cDNA of CruFc-1 consists of 523 bp with 405 bp CDS encoding a putative signal peptide of 17 amino acids and mature peptide of 117 amino acids. The full-length cDNA of CshFc consists of 433 bp with 315 bp CDS encoding a putative signal peptide of 17 amino acids and mature peptide of 87 amino acids. CruFc-1 has no introns. The promoter of CruFc-1 was cloned following as the method of genome walking. In order to verify the biological function of CruFc-1 and CshFc, the cDNA fragments encoding the mature peptides of CruFc-1 and CshFc were expressed in E. coli BL21(DE3)pLysS respectively. SDS-PAGE and Western blott revealed the production of recombinant CruFc-1 and CshFc proteins. The purified recombinant protein rCruFc-1 could effectively inhibit the growth of gram-positive bacteria. It played a key role for the 4-DSC domain of the crustin in inhibiting the growth of bacteria. The CruFc-1 was also expressed in Pichia pastoris and successfully obtained the recombinant protein in a 5-L fermenter. A full-length cDNA of ferritin (FcFer) was cloned from Chinese shrimp, F. chinensis. The full-length cDNA of FcFer consists of 1221 bp with a 510 bp ORF, encoding 170 amino acids. The deduced peptide has no signal peptide. The deduced amino acid sequence was highly homology to that of L. vannamei. Results of the RT-PCR showed that the expression of FcFer mRNA was prominently increased after shrimps were challenged with white spot syndrome virus (WSSV) in the laboratory and its expression was also increased when the shrimps were induced with heavy metal ions (Zn2+ and Cu2+). In addition, FcFer was successfully expressed in E. coli and purified with Co-NTA affinity chromatography. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments of the recombinant protein were identical to corresponding sequence of the ferritin from L. vannamei. On basis of the full-length cDNA deposited in the NCBI, MIH of F. chinensis was successfully expressed in E. coli and the interested protein was purified using the immobilized-metal affinity chromatography (IMAC). A single band was seen by urea-SDS-PAGE. The result of Western blot indicated that the recombinant protein expressed in E. coli could specifically react with the antibody of MIH from Metapenaeus ensis. Furthermore, in gel digestion and identification using LC-ESI-MS were used for peptide mass fingerprint (PMF). PMF queried against the NCBI database confirmed that the recombinant protein was identical to partial fragment of MIH from Marsupenaeus japonicus.