Dissertation > Excellent graduate degree dissertation topics show

Analysis the Major Adhesin Gene Sequences and It’s Recombinant Gene Products Function of Aeromonas Hydrophila

Author: WuXueMin
Tutor: LinTianLong
School: Fujian Agriculture and Forestry University
Course: Clinical Veterinary Medicine
Keywords: Aeromonas hydrophila Major adhesins Adhere to Suppression Blocking
CLC: S941
Type: Master's thesis
Year: 2010
Downloads: 31
Quote: 0
Read: Download Dissertation

Abstract

In order to thoroughly explore the differences as well as the main function of adhesins hydrophila Aeromonas (Ah) between different adhesin gene in the sequence aspects, to understand the major adhesins in invasive bacterial host-mediated host immune process may the role of of 6 Ah bacteria mainly adhesin gene cloning, sequencing, simultaneously Ah the bacteria ZN1 were main adhesin recombinant gene expression conditions were optimized, and part of the biological function of the expression product was Analysis to obtain the following results. The nucleotide and amino acid sequence analysis showed that: 6 Ah bacteria the adhesin gene nucleotide and amino acid homology rate ranges from 35.6% to 99.6% and from 34.2% to 99.5%; same source and the same serotype strains between BL ① BL ④ major adhesin gene nucleotide homology of up to 99.6%, corresponding to the amino acid homology of 84.6%; the main adhesion between different sources and different serotype strains BL (1) and TPS-30 the gene nucleotide homology of 98.1%, only 42.3% of the corresponding amino acid homology; strains ZN1 (serotype O: 10501, isolated from the European eel) the major adhesin amino acid sequence of other strains differences The greater homology only 34.2% to 63.6%. The results suggest that strains of Aeromonas hydrophila major adhesion gene nucleotide and amino acid homology with the source of strain and serotype no strict correlation, showing some diversity. The major adhesin gene recombinant expression conditions optimization results show that: ZN1 strains of Aeromonas hydrophila major adhesin gene (Mah) recombinant bacteria Ecoli BL21 (named ZAPB) enrichment 4hrs before induction and a 5hrs expression better than enrichment 2hrs, 3hrs; adding different inducer IPTG concentration (0.1 mmol / L, 0.5mmol / L, 1.0 mmol / L, 1.5 mmol / L) under conditions similar effect of expression; induced expression of 2hrs, 3hrs better than 4hrs 5hrs; under the conditions of 27 ℃, the inducible expression better than 30 ° C, 33 ° C and 37 ° C; In addition, the increase in oxygen flow, adjust the medium pH (6.0 and 8.0) and low temperature delay expression ( 16 ℃ induction 8h) can improve the effect of expression, and the expression product exists mainly in the form of inclusion bodies. The results showed that the optimum ZAPB expression conditions: shaking speed 200r/min, pre-enrichment induced 4hrs bacterial concentration OD600 = 0.6 ~ 0.8 between the concentration of the inducer IPTG 0.5mmol / L, induction temperature 27 ° C, The inducible expression 3hrs. The major adhesin recombinant expression product immunological characteristics and functional analysis of the results show that: (1) cross-ELISA results show that the rabbit anti-the gene donor the bacteria ZN1 strains natural outer membrane protein (ZN1-OMP) serum on the expression product Mah- TrxA (the formation of a fusion protein of thioredoxin with main adhesins) and the reaction of the Zn1-omp titers of 1:3200 and 1:12800; reaction of rabbit anti of Mah-TrxA serum Mah-TrxA and Zn1-omp titer of 1:12800 and 1:1600, respectively, indicating that the expression product and the natural outer membrane proteins have similar immunological properties. (2) The adhesion test the fish of sources Ah bacteria ZN1 strains and BL ① strain (serotype O: Ah10501), TPS-30 strain and Ah9805 strain (serotype O: 9) and CQ-3 strain (serotype O: CQ -1) relative carp epithelial tumor cells (EPC) adhesion rate of more than 97%; non-fish sources Ah ATCC7966 strains of bacteria (serotype O: 1) the relative adhesion of EPC was 78.5%. (3) adherence inhibition and blocking test display, EPC by Mah-TrxA preincubation adhesion test, strains with different Ah, the average EPC adhesion bacterial counts only 0.3 to 3.9 (lt; 10); tested different Ah strain of rabbit anti-serum Mah-TrxA preincubation with EPC adhesion test the average EPC adhesion number of bacteria is also only 0.3 to 3.7 (lt; 10). The results suggest that the Mah-TrxA different Ah strain cross-inhibition antigen competitive inhibition test and antibody blocking test could significantly reduce the of different Ah strain of EPC adhesion from both positive and negative aspects have prompted the cloning, expression Mah-TrxA indeed ZN1 strains main adhesins; also confirmed by means of genetic engineering techniques to the expression of the fusion protein expressed in the form of Mah-TrxA with wild strains ZN1 main adhesins have similar biological activity. In summary, the research topic analysis and comparison of six different Ah bacteria adhesin gene nucleotide and amino acid homology to clarify the Ah mainly adhesin gene homology source of strain and serotype no rigorous correlation; verified Mah-TrxA fish cell adhesion function, confirmed Mah ZN1 strain adhesin gene; cross inhibition test proved Mah-TrxA fusion proteins from different sources can be suppressed and serotype Ah strains of EPC adhesion, adhesion sites results prompted different Ah strain conservative. This finding may be able to fish hydrophila Aeromonas disease prevention and control provides a new way of thinking.

Related Dissertations

CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fish Diseases
© 2012 www.DissertationTopic.Net Mobile