Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacterin

A vaccine and protective antigen technology, applied in the field of bioengineering, can solve the problems of bacterial drug resistance, impact on application, large dosage, etc., and achieve the effect of low cost, easy operation and high yield

Inactive Publication Date: 2005-06-15
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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AI Technical Summary

Problems solved by technology

Traditional antibiotic treatment has played an important role in the prevention and treatment of bacterial Vibrio in seawater culture, but long-term use of antibiotics can cause bacterial resistance, and the dosage of drugs is getting larger and larger, resulting in drug residues and seriously affecting the quality of aquatic products. even threaten human health
In recent years, seawater Vibrio vaccines have developed rapidly and play an increasingly important role in the prevention and treatment of bacterial vibriosis, but they are mainly inactivated vaccines. Due to the presence of complex microbial metabolites in the preparation of inactivated vaccines, there may be Side effects, and the protective antigen may be destroyed in the inactivation process to affect its immune effect, and different serotypes lack cross-protection, which seriously affects its application
The new generation of genetically engineered subunit vaccines can solve the above problems. The first problem to be solved in the study of genetically engineered subunit vaccines is to find effective protective antigen genes. The outer membrane protein is a unique structure of the cell wall of Gram-negative bacteria. , located in the outermost layer of bacteria, has the opportunity of extensive contact with the body's immune system. Therefore, it has become a hotspot in the study of bacterial protective antigens. It plays an important role in being attacked by phage (FEMS Microbiology Letters 125 (1995) 101-106), but there is no relevant report on it as a protective antigen. The present invention carries out recombinant expression of the Ompk gene, and studies have found that its protein has a better Protective antigenic properties, can be used as a vaccine component to resist the invasion of Vibrio

Method used

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  • Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacterin
  • Expressing Ompk antigen of outer-membrane protein of Vibrio Harveyi and application as constituent of bacterin

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Embodiment 1

[0006] Cloning of embodiment 1 Ompk gene

[0007] PCR primers: primers were designed according to the published conserved sequence of the outer membrane protein Ompk gene of Vibrio species, primer 1 was 5'ATG CGT AAA TCACTT TTA GCT CTT AGC C 3'; primer 2 was 5'TTA CTG CGA TGT AGT GAC CGA AGC 3', synthesized by Shanghai Bioengineering Co., Ltd. Preparation of Ompk gene: using Vibrio harveyi EcGS020802 genomic DNA as a template, the target gene was obtained by PCR technique. The PCR cycle reaction conditions were: 94°C for 30s, 58°C for 30s, and 72°C for 90s for 30 cycles. Cloning and identification of PCR products: The purified foreign gene was connected with pGEM T vector, transformed into Escherichia coli, positive clones were screened by the blue-white method on LB solid medium containing ampicillin, and further identified by enzyme digestion. The above operations were carried out according to conventional methods. The positive clones were sequenced by ABI377 automatic seq...

Embodiment 2

[0009] Expression and purification of embodiment 2 recombinant Ompk gene

[0010] EcoRI and BamHI restriction sites were added to both ends of the mature peptide of Ompk gene, and the gene was modified by PCR method. The gene and vector pBV220 were respectively digested with EcoRI and BamHI, ligated at 4°C for 16 hours, transformed into Escherichia coli DH5α, and contained in Positive colonies were screened on LB plates with ampicillin, and plasmids were extracted for further enzyme digestion identification. The identified positive clones were cultured in liquid LB medium at 30°C. When the OD value reached 0.4-0.6, they were immediately induced by heat at 42°C. After induction for 4 hours, the bacteria were collected by centrifugation, ultrasonically disrupted, and inclusion bodies were collected. After the inclusion body is washed with 2-4M urea, it is lysed with 8M urea, and the residual urea is removed by water dialysis to obtain a recombinant protein with high purity. Suc...

Embodiment 3

[0011] Example 3 The immune control effect of recombinant protein on grouper Harvey vibriosis

[0012] 150 healthy grouper without disease, weighing 25-30g, were randomly divided into three groups, 50 in each group. Ompk recombinant protein immunization group was intraperitoneally injected with 50ug / tail antigen plus an equal volume of Freund's incomplete adjuvant (FCA). In the whole bacteria group, 0.1 ml of Vibrio harveyi EcGS020802 vaccine was injected into each tail. The control group was injected with normal saline 0.1ml per tail. 28 days after immunization, with LD 50 Dosage Vibrio harveyi Vibrio harveyi EcGS020802 was used to challenge and infect the three experimental groups, and the immune protection rates were calculated respectively. According to the one-way analysis of variance, the relative immune protection rate of the Ompk recombinant protein immunized group was 100%, and the immune protection rate of the whole bacteria group was 100%, both of which were sign...

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Abstract

An expression of the Hawei's vibrio outer membrane protein antigen Ompk in colibacillus and the application of the recombinant outer membrane protein Ompk as the component of vaccine in preventing the vibrosis of sea fish are disclosed. Said recombinant protein is prepared through providing Hawei's vibrio EcGS020802 DNA as template, cloning the coding gene of Ompk by PCR method, recombining the gene in the prokaryotic expression carrier pBV220, efficient expression of the recombinant protein in colibacillus to become inclusion body, washing, modifying and renaturation. Its advantages are high purity and high output.

Description

field of invention [0001] The invention belongs to the technical field of bioengineering, in particular to a DNA fragment and its expression product protein. The DNA fragment is recombinantly expressed in Escherichia coli, and the recombinant DNA molecule and its expression product can be used as a vaccine component against vibriosis. technical background [0002] Vibrio is a class of Gram-negative, polar flagellar, motile, non-spore-forming, short rod-shaped or arc-shaped bacteria that are distributed in coastal and estuary seawater and marine organisms, and are the most common bacteria in the marine environment One of the groups, some of which are pathogenic bacteria of marine organisms. As a pathogenic bacterium, Vibrio can infect a variety of farmed animals, such as shrimp, clams, abalone, fish, etc., often causing a large number of deaths and huge economic losses. Because they widely exist in natural sea areas and mariculture areas, vibriosis is prevalent worldwide an...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K39/395A61P31/04C07H21/00
Inventor 李宁求吴淑勤白俊杰劳海华石存斌叶星简清潘厚军陶家发
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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