| Accompanied by the traditional prevention and control methods cannot resolvethe frequent outbreak of aquatic animal diseases, the microbial prevention and controltechnology become the research hot spot. Probiotics has its unique advantage in theapplication of biofloc, bacillus being a major genus for probiotics, has becomepopular in scientists. But resent research of bacillus are commonly standard strains,therefore, host bacterium is waiting in bacillus species, bacillus study range isurgently to be expanded. Our laboratory separation bacterial isolates from intestine offarmed shrimp and biofloc in farming ponds, it has proven that some of them canenhance the ability of shrimp to resist the infection with white spot syndrome virus(WSSV).These isolates were firstly identified as Bacillus firmus, B. cereus, B. subtilis, B.baekryungensis, and B. aquimaris, respectively, with a systemic phylogenetic treeestablished based on their16S rDNA sequences along with46related sequences fromGenBank of NCBI. The BIOLOG analyzer was applied to conduct the analyses on theutilization of carbon sources. The results of susceptibility of these isolates to26antibiotics including ampicillin, kanamycin and chloramphenicol tested showed thatthe strain of B. firmus, B. subtilis, B. baekryungensis, and B. aquimaris are allsusceptible to ampicillin, kanamycin and chloramphenicol whereas the strain of B.cereus is susceptible only to chloramphenicol. Analysis on their natural plasmidsrevealed that all of the five wild isolates did not contain any natural plasmids. Basedon these result PC004,201112010603,201112010701,201112012401have thepotential to become the host bacteria.The flagellin gene of bacterial was used as molecular marker in its classificationand determination. In the study, Bhag primer of Bacillus subtilis was used toamplification Bacillus firmus hag gene, and1213bp product was obtained. Which isonly13%-15%similarity with Bacillus subtilis, and the otherness consist in thevariable region. Bfspecouter, Bfspecinnerprimer realized the nested PCR specificdetection of Bacillus firmus PC004and PC024, which designed based on the obtainedsequence. The specify detection method of Fluorescent Quantitation PCR establish with Bfspecinnerprimer. The limit detection of PC004is17.3CFU/ml, PC024is19.7CFU/ml. The specify detection method has strong specificity, cycle short and highsensitivity, provide technical assistance in practical application.Based on these results, plasmid pBE2was used as the exogenous plasmid totransform the five wild bacillus strains, respectively, via both protoplasttransformation and electroporation. The positive transformants were screened byusing both resistant plating and PCR verification. With protoplast transformationmethod201112010101,201112010603obtain positive trnsformant. For201112010101is not susceptible to ampicillin, the strain of201112010603was finallyselected as the potential host cells for plasmid pBE2.In order to realize the exogenous protein expression, plasmid pBE2wasreconstructed after it was transformed into B.subtilis. First,4412promoter and gfpgene come from pAD4412plasmid bonded into pBE2plasmid. Second, gfp gene wasplaced under the control of the P43promoter, which cloned from B.subtilis, wascloned into pBE2plasmid, and recombinant plasmid was designated pBE43GFP.Third, gfp gene was replaced by Cm gene, and recombinant plasmid was designatedpBE43Cm. Fourth, the hag promoter was applied instead of P43, and pBEhCmplasmid was recombinanted. Fivth, the recombinant plasmids were transformant intoB.subtilis vio protoplast method.The triple gene deletion in EvpC, EsrB and PstC of Edwardsiella tarda is used asthe host bacteria. Plasmid pBAD-gIIIA contains VP28gene is transformed intoE.tarda by electroporation.0.02%L-arabinose is used to induce the VP28gene,SDS-PAGE, Western Blotting and proteome analysis methods result shows that theVP28gene is sucessfuly expressed in E.tarda. The espressed bacterial coated foodpellets to test and verify the preventive effect. |
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