| Cyclina sinensis are important marine economic animal in China,given its high nutritional value and delicious taste,so Cyclina sinensis in aquaculture industry has the important status.But in recent years,due to domestic coastal areas to take the bait is cast and high density,high yield cultivation way,make the germplasm of Cyclina sinensis serious recession,disease resistance decline gradually,combined with the cultivation of regional water pollution is serious,year after year in the breeding of Cyclina sinensis diseases and deaths.This study trough the Cyclina sinensis immune related factor to study the expression of serine protease inhibitors,the immune mechanism was revealed from the molecular level and signal transduction,to explore Cyclina sinensis immune disease-resistant mechanism,establishing the clam health cultivation way provide valuable theoretical basis and experimental data.This paper introduces the Cyclina sinensis economic value and research background,this paper expounds the current common problems and the reasons of shellfish aquaculture.Especially on the pathogenic microorganisms-vibrio anguillarum the harm of Cyclina sinensis breeding,and analyzes the pathogenesis and prevention and cure of vibrio anguillarum.It also introduces the serine protease inhibitor gene structure and its important role in shellfish immune regulation.Researchers constructed by the method of high-throughput sequencing SMART cDNA library,using bioinformatics analysis method,the Cyclina sinensis serine proteinase inhibitor(SPI)length of the gene sequence.Results show that the SPI gene is 587 bp,open reading frame is 456 bp,encoding 151 amino acids,its molecular weight was 16.850 KD protein theory,theory of isoelectric point pI=5.74,in addition,the amino acid sequence 1-19 for the signal peptide sequence,48-88 and 111-148 for KAZAL region.The highest(13.2%)amino acid composition is glycine(Gly).With KAZAL other family members to homology comparison found that the gene sequences of the family of KAZAL rich in cysteine.Because the animals cysteine has detoxification,maintain normal metabolism,improve inflammation and allergic reaction,adjust the organism itself defense mechanisms,and other functions,so the SPI gene plays an extremely important role in immune reaction process.In the phylogenetic tree showed that Cyclina sinensis Kazal serine proteinase inhibitor and the evolution of the mollusks Kazal serine proteinase inhibitor distance is relatively close,and the distance of vertebrate evolution.This study also used real-time fluorescence quantitative rt-pcr method,determination of the SPI gene in Cyclina sinensis shell muscle,liver,blood and closing coat film and gill of five different tissue distribution as well as the gene in the blood after vibrio anguillarum stimulation sequence sexual expression.Results showed that SPI genes in Cyclina sinensis are expressed in the above five groups,but the expression difference obvious,which express the highest levels in the blood,is significantly higher than other groups(P<0.05),closed shell express times muscle,liver,minimum content of coat film and gill,show that SPI gene expression mainly in the blood.In addition,after the vibrio anguillarum infection Cyclina sinensis,eyes only for beta actin gene,using real-time fluorescent quantitative PCR analysis SPI genes expressed in the scheduling in the blood.The results showed that the experimental group after infection 3 h express a significant rise in quantity,and extremely significant difference with the control group(P<0.01);And in 3 h express maximum amount,about about three times in the control group;At 6 h the expression quantity decreased,but still significantly higher than control group(P<0.05);Within 24 h and 48 h a significant rise in its expression quantity,and expressed in 48 h and reached a peak and extremely significant difference with the control group(P<0.01);9 h,12 h and 96 h gene expression and the control group difference is not obvious,that SPI plays a very important role in the immune genes.Researchers use the method of genetic engineering to establish prokaryotic expression system,use the SPI genes connected with pET-30a expression vector and transformed into the Escherichia coli strain BL21(DE3)pLysS,the fusion protein reached a maximum 4h later after induced by lmmol/LIPTG SDS-PAGE indicated that the fusion protein molecular weight is 23KDa agreed with expectation.consistent with expected protein molecular weight,by affinity chromatography and dialysis method and renaturation method,obtain the recombinant protein with biological activity.At the end of the paper studied the serine protease inhibitors of antibacterial effect,use of recombinant protein powder is detemined by coomassie brilliant blue method after PBS solution concentration,use of Kazal serine protease inhibitor proteinase reaction analysis kit,recombinant protein activity concentrations were reveals the in vitro antibacterial activity of recombinant proteins and immune regulating function in the body.,according to the results of freeze-dried recombinant protein after 1 MLPBS buffer dissolved,its concentration is 2.576 g/l.After determination of enzyme reaction analysis,the energy density of about 435.1667 IU/L.The in vitro bacteriostasis experiment that the recombinant protein has obvious antibacterial activity in vitro,can directly inhibit the growth of vibrio anguillarum in vitro.Mix recombinant protein with vibrio anguillarum incubation after injection into Cyclina sinensis,compared with the pure injection of vibrio anguillarum,experimental group Cyclina sinensis showed stronger microbial removal efficiency,obviously the recombinant protein can enhance Cyclina sinensis immune prevention ability,explain the inside the Cyclina sinensis played an important role in immune disease-resistant.In conclusion,this study first found the Kazal serine proteinase inhibitor gene in Cyclina sinensis,and analyzes the molecular structure characteristics and physical properties such as isoelectric point to study the expression change after the stimulation and the in vivo and in vitro antibacterial activity,further clarify the Cyclina sinensis path of immune recognition and immune regulation mechanism provides an important experimental data. |
PDF Full Text Request