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Cloning and Expression of Two Aeromonas Strains Outer Membrane Protains and Chinese Softshell Turtle IL-8

Author: ZhangLeDai
Tutor: ZhaoBaoHua
School: Hebei Normal
Course: Microbiology
Keywords: Chinese softshell turtle Aeromonas hydrophila Aeromonas veronii outermembrane protein transient expression plant vaccine interleukin8 chemotactic activity
CLC: S947.1
Type: Master's thesis
Year: 2012
Downloads: 30
Quote: 0
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Abstract


Chinese softshell turtle(Pelodiscus sinensis or Trionyx sinensis) is an importanteconomic aquatic product in our country, which is favored broadly by the domestic andforeign consumers with its delicious meat quality and rich nutrition. But in recent years,Chinese softshell turtle pathogenic diseases has broken out heavily and caused heavy losses inChinese softshell turtle breeding industry of our country. Therefore, the prevention andtreatment of Chinese softshell turtle pathogenic diseases have become the primary problem inChinese softshell turtle breeding industry of our country.This paper studied the prevention and treatment of Chinese softshell turtle pathogenicdiseases by the way of specific immunity and nonspecific immunity. In the way of specificimmunity, the outer membrane protain A genes of Chinese softshell turtle pathogensAeromonas hydrophila HBNUAh01and Aeromonas veronii HBJY01had been cloned, and itsexpressions in tobacco (Nicotiana tabacum) leaf cells had been detected. A. hydrophila outermembrane protein A (AhompA) gene and A. veronii outer membrane protein A II (AvompA II)gene were amplified by PCR using A. hydrophila HBNUAh01cells and Aeromonas veroniiHBJY01cells as templates respectively, and separately cloned into the pEASY-Blunt Simplevector for sequencing. After sequence confirmation, AhompA gene and AvompA II gene wereinserted into expression vector pCAMBIA1300respectively, which contains a yellowfluorescent protein (YFP) gene, to obtain recombinant expression plasmids. The recombinantexpression plasmids were introduced into Agrobacterium tumefaciens GV3101competentcells, and the positive clones were transfected to tobacco leaf cells. The expressions of yellowfluorescent proteins separately fused with AhompA and AvompA II were observed byConfocal Laser Scanning Microscope, and the mRNA of AhompA and AvompA II wasdetected by RT-PCR. The AhompA gene cloned from A. hydrophila HBNUAh01was1032bp,the AvompA II gene cloned from A. veronii HBJY01was996bp. The fusion proteins of YFPseparately fused with AhompA and AvompA II were successfully expressed in tobacco leafcells. The successful expressions of AhompA gene and AvompA II gene in tobacco leaf cells have laid a foundation for further investigation of prevention of limnobios diseases caused byA. hydrophila and A. veronii with plant vaccine.In the way of nonspecific immunity, the mature peptide gene of Chinese softshell turtleinterleukin8(Psil-8(m)) and the precursor peptide gene of Chinese softshell turtle interleukin8(Psil-8) had been respectively cloned from Chinese softshell turtle liver tissue and expressedin E.coli expression system. Then, after analysis and purification, confirmed the proteinfunction by chemotaxis test with Chinese softshell turtle neutrophils. The location of signalpeptide of Chinese softshell turtle interleukin8protein sequence was analyzed with the signalpeptide analysis software Signal P3.0. According to the result, specific primers were designedto amplify the Psil-8(m) gene and Psil-8gene separately from Chinese softshell turtle livertissue. And these two genes were cloned into the pMD19-T Simple vector respectively forsequencing. After sequence confirmation, Psil-8(m) gene and Psil-8gene were inserted intoexpression vector pET-25b separately to obtain recombinant expression plasmids. Therecombinant expression plasmids were introduced into E.coli BL21(DE3) competent cells.After SDS-PAGE analysis of the IPTG induced expression of the positive clones, the interestproteins were purified via Ni-NTA and analyzed by Western Blot. Then confirmed thefunctions of purified proteins by chemotaxis test with Chinese softshell turtle neutrophils. ThePsil-8(m) gene cloned from Chinese softshell turtle liver tissue was246bp and the Psil-8gene was312bp. The interest proteins of Psil-8(m) gene and Psil-8gene were successfullyexpressed in E.coli BL21(DE3). The CI(chemotactic index) of both Psil-8(m) and Psil-8toChinese softshell turtle neutrophils were the highest, when the protein levels between0.9-1.35μg/μL. The maximum CI of Psil-8(m) was3.710±0.334, the maximum CI of Psil-8was2.634±0.390. And the CI of both Psil-8(m) and Psil-8showed decreasing trend, when theprotein concentration exceeded1.35μg/μL or was lower than0.9μg/μL. The successfulexpressions of the Psil-8(m) and Psil-8and successful confirmations of their chemotacticactivities have laid a foundation for prevention and treatment of Chinese softshell turtlepathogenic diseases by the way of nonspecific immunity.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Other aquatic pests and diseases and their prevention > Turtle
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