Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Construction and characterization of invasion protein B gene deleted mutant asd host-vector balanced lethal system / 中国免疫学杂志

Objective: To develop an oral live vaccine vector which stably carries exogenous genes.Methods:SL1344ΔsipBΔasd host-vector balanced lethal system was constructed by the method of recombinant suicide plasmid-mediated allelic exchange on the basis of attenuated Salmonella typhinurium SL1344ΔsipB.Then,the biological characteristics of SL1344ΔsipBΔasd was analyzed.Results:The results showed that the mutant was stabile with the Δasd gene in vitro;the serotype and growth rate of SL1344ΔsipBΔasd strain was almost same as the parent SL1344ΔsipB and SL1344 strain.And the mutant strains remain swim ming zones.Virulence test in mice showed that the virulence of SL1344ΔsipBΔasd which carried complementary plasmid pYA3493 by electro-transformation decreased by 1.4%compared with SL1344.Conclusion: These results showed that the SL1344ΔsipBΔasd mutant was successfully constructed.It is likely that this mutant should be used as a live vector to express foreign genes.

Construction and characterization of host-vector balanced lethal system of attenuated salmonella choleraesuisΔcrpΔcyaΔasdC78-1 (pYA3493) / 中国免疫学杂志

Objective:In order to develop an oral live vaccine vector of swine that can stably carry exogenous genes.Methods:Mutant ΔcrpΔcyaΔasdC78-1 was constructed by the method of suicide plasmid pREasd-mediated bacteria homologous recombination on the basis of attenuated Salmonella choleraesuisΔcrpΔcyaC78-1.Complementary plasmid pYA3493 with asd was electrotransformed into the mutant,and thenΔcrpΔcyaΔasdC78-1(pYA3493) host-vector balanced lethal system was constructed.Its biological characteristics were analyzed further.Results:The results of PCR and sequencing showed thatΔcrpΔcyaΔasdC78-1(pYA3493) was constructed suc-cessfully.Biological characteristics showed that the serotype of ΔcrpΔcyaΔasdC78-1(pYA3493) was identical to ΔcyaΔasdC78-1 and vaccine strain C500 and it can stably carry theΔasd gene in vitro;its growth speed was a little slower than ΔcrpΔcyaC78-1 strain,but both of their growth speeds were significantly slower than vaccine strain C500;the biochemical characteristics of ΔcrpΔcyaΔasdC78-1 ( pYA3493 ) were basically the same with ΔcrpΔcyaC78-1 strain.Oral virulence test in mice showed that the virulence ofΔcrpΔcyaΔasdC78-1 ( pYA3493 ) was similar with ΔcrpΔcyaC78-1, but its median lethal dose is 412 times of vaccine strain C500.Conclusion:These results demonstrated that attenuated Salmonella choleraesuisΔcrpΔcyaΔasdC78-1(pYA3493) strain had the potential to be used as an oral live vaccine vector for expressing foreign genes efficiently.

Construction and characterization of invasion protein B gene deleted mutant of Salmonella typhimurium SL1344 strain / 中国免疫学杂志

Objective:In order to develop a safer vaccine strain exploit Salmonella typhimurium vaccine strain .A ΔsipB mutant of Salmonella typhimurium SL 1344 strain was constructed.Methods: Firstly, the recombinant suicide plasmid containing the missing 585 bp sipB ( PREΔsipB ) was built by homologous recombination , and screenned by two-step method.Results: PCR and sequencing results showed that SL 1344ΔsipB was successfully constructed.It was no significant changes compared with SL 1344.But compared with the parent strains SL 1344 , the mutant strain had obvious change in its virulence , oral challenge of bacteria in mice revealed that LD50 of the mutant strain was 1.70 ×108 CFU,the toxicity reduced about 1.4%.The protection rate induced by the sipB mutant was 50%,and the serum antibody peaked 14 d post-immunization.Conclusion:The SL1344ΔsipB mutant was constructed suc-cessfully,and genetic stability ,significantly reduced virulence.The study provides a new approach for further study of the relationship between the gene and pathogenicity of Salmonella typhimurium.It is likely that the ΔsipB mutant could be adapted to develope attenuated Salmonella vaccine.

Construction of Salmonella Pullorum C79-13ΔcrpΔasd mutant balanced-lethal host-vector system and its biological features / 中国免疫学杂志

In order to develop a safer vaccine strain exploit Salmonella Pullorum vaccine strain as a live vaccine vector.Methods:AΔcrpΔasd mutant of S.pullorum C79-13 strain was constructed and it was developed E.coli donor strain mutant was generated through the two-step method introduced by χ7213 ( pREΔasd) was conjugated with the recipient of C 79-13Δcrp.The C79-13ΔcrpΔasd the transduction of recombinant suicide plasmid .Results:PCR and sequencing results showed that ΔsipBSL1344 was suc-cessfully constructed.The further study indicated that foreign DAP must be supplied for the ΔcrpΔasd mutant to grow,in addition,the asd gene was transmitted stably .Compared with C79-13 strain,the O antigens was identical to C79-13 strain,but the growth velocity was reduced significantly ,significantly reduced virulence .Conclusion: The ΔcrpΔasd mutant can accept asd+plasmid to construct host-vector balance lethal system , and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.

Expression and characterization of the dermonecrotic toxin gene of Bordetella bronchiseptica / 生物工程学报

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.

Influence of fusion protein of IBDV VP2 and chicken interleukin-2 on immune response in chicken / 生物工程学报

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.

Characterization of attenuated Salmonella C500 strain with a delta asd mutant and use as an Asd+ balanced-lethal host-vector system / 生物工程学报

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a delta asd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis delta asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of delta asdC500 mutant, the vector control delta asdC500 (pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of delta asdC500 mutant, delta asdC500 (pYA3493) and delta asdC500 (pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of delta asdC500 (pYA3493) or the parent C500 strain, and the virulence of delta asdC500 (pYA3493) with LD50 of 1.1 x 10(7) CFU was a little lower than C500 with LD50 of 4.4 x 10(6) CFU. All piglets inoculated with delta asdC500 (pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant delta asdC500 (pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the delta asdC500 mutant had a series of biological characteristics similar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system.