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Construction and Immunogenicity of an △apxIC/ompP2 Mutant of Actinobacillus Pleuropneumoniae Serovar 5 Gomphosised ompP2 Gene of Haemophilus Parasuis Serovar 5

Author: GongYuHeng
Tutor: WenXinTian；CaoSanJie
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: APP Hps ApxIC ompP2 Biological characteristics Immunogenic
CLC: S852.61
Type: Master's thesis
Year: 2011
Downloads: 34
Quote: 1
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Abstract


Pleuropneumoniae Actinobacillus (Actinobacillus pleuropneumoniae APP) is a herd highly contagious respiratory infectious disease pathogens, can trigger pleuropneumoniae (Porcine Contagious pleuropneumonia, PCP); the Haemophilus parasuis (Haemophiius parasuis HPS) colonize the upper respiratory tract in pigs normal flora and opportunities of pathogens can cause herd of Haemophilus parasuis disease, these two epidemics of serious harm to the development of pig industry. Currently to build APP and of Hps two pathogens immunogen gene attenuated deletion mutant has become an important research direction of both diseases of new vaccines. In this experiment, the APP serotype 5 bacteria as the parent strain, constructed chimeric HPS serotype 5 HS80 ompP2 strains gene APP ApxIC gene deletion mutant and its biological characteristics and immunogenicity. 1, fitting Hps APP gene deletion mutant recombinant transfer vector construct: the middle of the recombinant transfer vector pBOSK △ IC-1 Construction: the gene downstream 1434bp sequence amplification ApxIC APP serotype 5 SC-1 strains genome as a template for the right homology arms added in its upstream the SpeI and M1 UI sites, the amplified fragment TA-cloned into pMD19-T Simple, a 1400 bp fragment obtained by PCR by double digestion correctly. Double digestion for target fragment and use its replacement the original pBOSK △ IC right homology arms by PCR a 1400 bp fragment, 1400 bp and 7000bp fragment obtained by double digestion, that the test transformation vector pBOSK △ IC, swim in the right homologous arm into the MluI restriction sites successfully constructed recombinant transfer intermediate carrier pBOSK △ IC-1 recombinant transfer vector pBOSK △ IC/ompP2 build: Hps blood type 5 HS80 strain genome as a template the amplification ompP2 gene fragment 1107bp sequence of the amplified fragment TA-cloned into pMDl9-T Simple identified by PCR a fragment about 1100bp, and the double digestion and sequencing are correct. The purpose of double-digested fragment connected to the middle of the recombinant transfer vector pBOSK △ IC-1, one about 1100bp fragment obtained by PCR, two double digestion were 1100bp and 8500bp bands to prove that the test 1107bp of HPS ompP2 gene inserted into the middle of the recombinant transfer vector pBOSK △ IC-1, successfully constructed chimeric the HPS ompP2 gene recombinant transfer vector pBOSK △ IC/ompP2.2, constructed chimeric HPS gene APP gene deletion mutant: The recombinant transfer vector pBOSK the △ IC/ompP2 electrically transferred to the APP serotype 5 Competent SC-1, the first homologous recombination, having a kanamycin-resistant colonies were screened from the TSA plates containing kanamycin, PCR identified two were about 1400bp and 800bp bands by the resistance experiments show the correct transfer of strain TSA plate containing sucrose, the second homologous recombination, screening sucrose resistant colonies do PCR identification give a Treaty prove that the test successfully screened a chimeric the HPS ompP2 gene APP ApxIC gene deletion mutants, named △ apxIC/ompP2.3 mutant strains to △ apxIC/ompP2 the biological characteristics and immunogenicity of a 1400 bp fragment, : growth characteristics test confirmed the mutant strains to △ apxIC/ompP2 the growth of the parent strain there was no significant difference; genetic stability test confirmed that the gene can stably inherited in vitro for 10 passages; hemolysis test confirmed in vitro does not show hemolytic activity ; cytotoxicity test confirmed its cytotoxic than the parent strain weakened; LDS lethal dose by half of the mice. Determination of mutant to 1.0 × 107CFU, the parent strain of 3.5 × 105CFU, a difference of 30 times, significantly reduce the toxicity of mice; the in protective immunity test, immunized mutant mice 20 times LDs. The parent strain to provide 100% protection, protection of LDso 5 times HPS HS80 62.5%; confirmed by ELISA for detection of immune serum of mice induced by antibodies to APP and HPS, and three free antibody concentration to achieve the highest. These trials showed that significantly reduce the virulence of the mutant APP infection compared with comprehensive, effective protection at the same the Hps infection also showed some protection Save, as for the further study of the mutant strains the practical application of the foundation.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
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