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Construction and Expression of Vibrio Vulnificus vvhA Cytolysin Genes

Author: LiYongTao
Tutor: LuZuoQuan
School: Zhejiang University
Course: Pathogen Biology
Keywords: Vibrio vulnificus Trauma vvhA gene Base sequence Clone Molecular vvhA fusion protein
CLC: R346
Type: Master's thesis
Year: 2004
Downloads: 75
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Abstract

Vibrio vulnificus (Vibrio vulnificus) is an encapsulated gram-negative halophilic Vibrio exist in seawater and seafood, equal to the first reported in 1979 by Farmer. The bacteria are the main cause of primary septicemia and severe wound infection. Sepsis and more associated with eating raw oysters and other shellfish and seafood bacterial particular, occur in cirrhosis, hemochromatosis, thalassemia charge the amount of iron in the body too much crowd, the case fatality rate of more than 50%. Skin wound infection by Vibrio vulnificus in seawater, which often leads to severe tissue necrosis. Vibrio vulnificus exact pathogenic mechanism has not yet been fully understood. Cytolysin by the structural gene vvhA encoding a molecular weight of the extracellular protein of 50,851 Da, and is unstable to heat, can be dissolved mammalian erythrocytes and Chinese hamster ovary (CHO) cells, Vibrio vulnificus to the extracellular release of the sole cytotoxic, has long been considered an important virulence factor of Vibrio vulnificus. The this article amplified full-length vvhA gene from the Vibrio vulnificus standard strains GTC333 in high fidelity PCR, TA cloning and sequencing construct prokaryotic expression system pET32a () the-vvhA-BL21DE3 fusion protein expression with different concentrations of IPTG induction vvhA, SDS- The PAGE analysis vvhA converged protein molecular weight. Its product as Vibrio vulnificus antigen genetically engineered vaccines and diagnostic kits. Experimental Method 1. Vibrio vulnificus culture: donated by the Department of Microbiology Department of the University of Gifu, Japan Vibrio vulnificus standard strains GTC333 strains coating on blood agar medium in aerobic conditions at 37 ℃ overnight, see a small white colonies around completely hemolytic ring, Gram stain negative small Vibrio automated analyzer test Zhejiang University Affiliated Children's Hospital laboratory bacteria Vibrio vulnificus. 2. Vibrio vulnificus template DNA preparation: using the conventional phenol - chloroform extraction the Vibrio vulnificus GTC333 strains of DNA by DNA enzyme, RNA enzyme eliminate Journal of Zhejiang University Li Yongtao again extracted with phenol-chloroform method of DNA dissolved in TE buffer, DNA concentration and purity were determined by spectrophotometry. 3.PCR amplification primers were synthesized by Shanghai Boya Biotechnology Co., Ltd. (BioAsia): According to the trauma of Vibrio standard strains GTC333vvhA reference nuclear battalion acid sequence and expression vector cloning sites endonuclease map analysis results, design their own. vv teams gene primer sequences are as follows: the upstream 5 the benevolence GceG once the fish workers Dandan ATGAAGAAAATGAeTeTG 3 / (BamHI) downstream 5 the benevolence GeeAAGeTTeTAGAGTTTGAeTTGTTG a 3 / (Hind Hill). Shanghai gaming of the Biotech Co., Ltd. (BBST) Pfu Taq mixed-fidelity PCR kit amplified vvhA gene PCR total reaction volume of 100 Lin 1, primer concentration of 250 nmol / L, 100 ng of DNA template. PCR parameters: 94oC5min, xl; 94 ° C 305,52 oC3OS 72oCgOS X 10; with 94oC3OS, 52oC305, 72oCIOOS (later increased per cycle 105), x25; at 72 ° C for 10min, xl. A 1.5% agarose gel electrophoresis to detect amplification products. 4. Ding A cloning, sequencing and expression vector construction: T-A cloning kit using BBST company will be amplified by the purpose of the cloned to pUCm T vector, transformed in E.Col 1 DH SQ strains and amplified. alkaline denaturation method of extracting plasmid. Satisfactory sequencing results, were amplified the containing pUCm a T vvhA and pET32a () E.eoli the DHS. After the plasmid was extracted target gene fragment was obtained by double digestion with pET32a () connection is transformed into the E.coli BL21DE3, amplification, plasmids were extracted again after sequencing, nuclear operators acid sequence with the reported vvhA gene for homology comparison. 5 Expression and purification of recombinant proteins: pET32a () a vvhA a BL21DE3 LB medium containing respectively 1.0, 0.5 and 0.1 mmol / L IPTG shaking culture, inducing a temperature of 37 ° C; IPTG induction product using ultrasonic crushing S000r / min4 ℃ were centrifuged for 10 min the supernatant and precipitation; 10% SDS-PAGE check the expression of the target protein. NTA affinity chromatography (BBST) to collect the expression of the target protein. The results of PCR from the Vibrio vulnificus the standard strain GTC333 strain DNA template expected size of vv amplified bands. Zhejiang Master Bu Dissertation Li Yongtao 2. Nuclear ordinary acid sequence analysis: Vibrio vulnificus in the pUCm a T with pET32a BU) recombinant plasmid the standard strain GTC333 strains vvhA gene nuclear acid sequence is identical. Nuclear battalion acid sequence homology compared with has reported the vvhA sequence 95.970rk, the deduced amino acid sequence homology to 98.51% .3. Purpose of fusion protein expression: 1.0, 0.5 and 0.1 mmol / L workers PTG well induce the expression of recombinant vvhA expression product after the precipitate the crushing was centrifuged sonicating, the expression of the amount of about 30% to 40% of the total bacterial proteins. Conclusion In this study, the cloning of Vibrio vulnificus the standard strain GTC333 strains, the hA the gene nuclear celecoxib acid sequence comparison with the reported corresponding sequences the nuclear battalion acid sequence homology of 95.97%, 98.51% amino acid sequence homology, the results show, we successfully amplified from genomic DNA of Vibrio vulnificus and cloned vvhA gene. Construction of this experiment pET32a () the one vvhA BL21DE3 system in low IPTG concentration (0.1mm. 1 / L) can also be a good induction expression of recombinant vvllA, its expression level as high as the total bacterial proteins 3 both 40? around, and mainly in the form of inclusion bodies. The experiment successfully constructed from standard strains of Vibrio vulnificus vvhA efficient prokaryotic expression system antigen to prepare for further study of the pathogenic mechanisms and immunodiagnostic kits.

Construction and Expression of Vibrio Vulnificus vvhA Cytolysin Genes

Abstract

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