[Clone, construct, expression and verification of lactoferricin B gene and several sequence mutations in yeast].

OBJECTIVE: To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary. METHODS: By self-template PCR method, the gene of Lactoferricin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities. RESULTS: The PCR amplifying and DNA sequencing tests indicated that the purpose plasmid contained the Lactoferricin B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary. CONCLUSION: The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae; the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.

[Application of M-RAPD technique to obtain the genomic fingerprints of various pathogenic microbials].

OBJECTIVE: To obtain genomic fingerprints of different pathogenic microbials and make certain whether their patterns can be used in the identification of microbials by means of multiplex random amplified polymorphic DNA analysis (M-RAPD). METHODS: Arbitrary primers of 10 oligonucleotides were randomly grouped, and various microbials chromosomal DNA were amplified with three combinatorial primers at a special higher annealing temperature. The products were detected by 15 g/L agarose electrophoresis and the patterns were analyzed by the software of Gelworks 1d Intermediate. RESULTS: Specific and resistant DNA fingerprints for different pathogenic microbials with combinatorial primers were gained. The profiles were clear, well-distributed and the number was great. The products of the three primers included most products of every two primers and would appear with no relation to their length, but small products had more opportunity; three primers could provide information contents half as many again as that two primers could provide for the same pathogenic microbials. There were obvious differences among different drug-resistant strains and between the drug-resistant strains and the corresponding reference strains, but different strains of the same microorganism had more similarity than discrepancy. The analytic data of the software of Gelworks 1d Intermediate also support our results. CONCLUSION: M-RAPD is a simple and rapid technique for the identification of different kinds of pathogenic microbials, and it can provide rich genetic information.

[Cloning the gene fragment coding the putative integrase-like protein from X maltophilia].

OBJECTIVE: To amplify the nucleotide sequence of CG-like receptor from X anthomonas maltophilia(X maltophilia). METHODS: Using the specific primer P1 designed by Grover's reported 342 bp partial nucleotide sequence of X maltophilia CG-like receptor and random primer to PCR amplify, PCR product was cloned in the pUCm-T vector. After the recombinant plasmid was tested by restriction endonuclease digestion, the insert on the recombinant plasmid was sequenced and analyzed. RESULTS: About 500 bp PCR product was cloned in the pUCm-T vector and obtained the recombinant pUCm-Int. By sequencing to the insert on the pUCm-Int with M13 universal sequencing primers, the 410-486 bp fragment of the cloned 510 bp nucleotide sequence (GenBank accession number: AY363962) showed 84% identity with the 9304-8958 bp fragment of the XACb0009 gene on plasmid pXAC64 of Xanthomonas axonopodis pv. citri. And the 4-166aa fragment of its translated 169aa sequence had 62% identity with the 38-200aa sequence of integrase-like protein coded by the XACb0009 gene. CONCLUSION: The cloned 510 bp nucleotide sequence was possibly the partial gene sequence coding the integrase-like protein of X maltophilia.