Dissertation > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fish Diseases > Microbial fish diseases

Cloning, Prokaryotic Expression of CpsD Gene of Streptococcus Iniae and Its Application on Indirect in Situ PCR

Author WangHaoZuo
Tutor WangKaiZuo
School Sichuan Agricultural University
Course Basic Veterinary Science
Keywords Streptococcus iniae cpsD gene Molecular characterization Cloning Prokaryotic expression indirect in situ PCR
CLC S941.4
Type Master's thesis
Year 2013
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The first isolation of Streptococcus iniae was Inia geoffrensis in1976. Not only has it been a threat to the worldwide aquaculture industry, but also a pathogen to human in the past years. It is generally believed that S. iniae is a major cause of serious bacterial infections in both fish and human in the world, making more and more people arise the interest and forcus on its research. The S. iniae strain DGX07(GenBank NO:FJ951434) was isolated from infected channel catfish. The molecular characterization, prokaryotic expression of cpsD functional gene of S.iniae and its further application on indirect in situ PCR were subsequently estabulished. The study was to provide some basic insights of characteristics of cpsD gene of S. iniae and support molecular biological characteristics of the pathogen and pathogenic mechanism to additional resources. It lays a stable foundation for the development of vaccine.1. Clonings identification and molecular characterization of cpsD gene of S. iniaeCloning and identification of cpsD gene of S. iniae isolate were performed, meanwhile, Molecular characterization of cpsD gene of S. iniae was analyzed by using bioinformatics softwares. The cpsD gene was720bp in length and contained a complete ORF, encoding a polypeptide of239amino acid and contained a named P-loop NTPase domain superfamily. CpsD Protein was highly conserved and has100%homologys among other S.iniae strains. The predicted results by bioinformatic softwares indicated that the hydrophobic regions were larger than hydrophilic regions; the putative protein had no signal sequence cleavage site or transmembrane domain, and had one potential glycosylation sites and twenty-two phosphorylation sites. The codon usage bias analysis of cpsD gene showed that the cpsD gene have strong bias towards A-ended or T-ended codon at the third codon position; compared to in Escherichia coli (E. coli), yeast and human, which has significant different were33,26and31, respectively.2. Prokaryotic expression and polyclonal antibody preparation of cpsD gene of S. iniae Recombinant expression plasmid pET-32a(+)-cpsD was constructed and then was was transformed into E. coli BL21(DE3)/pLysS competent cell, which is expressed through IPTG induction. SDS-PAGE showed a specific band of approximately46kDa, expressed in the insoluble fraction, as forms of lbs. The optimal growth condition for expression of CpsD was37℃for5h with0.6mM IPTG. The rabbit was immunized with the purified CpsD protein, The purified anti-serum reacted with the recombinant protein by Western Blot analysis. The agar gel diffusion test showed that the titer of prepared polyclonal antibody was1:16.3. Distribution pattern of S. iniae isolates in channel catfishChannel catfish were artificially infected by intraperitoneal injection with S. iniae isolates(LD50). cpsD gene of S. iniae was used to design a pair of specific primers and an oligonucleotide probe. Indirect in situ polymerase chain reaction were performed to detect bacteria in tissue distribution. The results showed that the positive signals sporadically appeared in the liver and the spleen after4hours after injection; After8hours, the kidney was detected positive signals as well;24hours later, positive signals were found in gill、heart、eyeball and brain, too. Lots of positive spots were detected in liver, spleen, kidney.

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