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Method for preparing bacterial ghost vaccine of haemophilus parasuis as well as product and application thereof

A technology of Haemophilus suis and germs, applied in the preparation of Haemophilus parasuis shadow vaccine and its products and application fields, achieving the effects of high lysis efficiency, large-scale production capacity, high lysis efficiency, and high initial induction concentration

Inactive Publication Date: 2013-12-18
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More critically, the cleavage vector pBV-Eprom constructed based on the mutated E gene Eprom can use a fermenter to prepare a large number of bacteria ghosts of Haemophilus parasuis, making large-scale production possible, but there is no correlation in this aspect research report

Method used

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  • Method for preparing bacterial ghost vaccine of haemophilus parasuis as well as product and application thereof
  • Method for preparing bacterial ghost vaccine of haemophilus parasuis as well as product and application thereof
  • Method for preparing bacterial ghost vaccine of haemophilus parasuis as well as product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of the phage lytic gene of embodiment 1 mutation

[0037] Primers were designed according to the coding sequence of phage PhiX174 cleavage gene E in GenBank:

[0038] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO. 2) Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO. 3)

[0039] Restriction sites EcoR I and BamH I were introduced into the 5' ends of the upstream and downstream primers, which were synthesized by Harbin Boshi. Use bacteriophage PhiX174 double-stranded DNA as a template to amplify cleavage gene E: PCR amplification reaction system is 50 μL, in which MgSO 4 2mM, 1μM each for upstream and downstream primers, dNTP200μM, 10x Taq buffer, Taq TM DNA polymerase 2U (TaKaRa), template DNA 10ng. The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 30s, 59°C for 30s, 72°C for 30s, and 72°C for 5 minutes. The E gene amplified by PCR is recovered by gel electrophoresis, and then the E gene is digested w...

Embodiment 2

[0041] The construction of embodiment 2 cracking plasmid vector pBV-Eprom and the screening of culture conditions

[0042] After purification of the cleavage gene E cloned in Example 1, it was digested with EcoR I / BamH I, then ligated with the pBV220 vector digested by EcoR I and BamH I with T4 DNA ligase (TaKaRa), and ligated overnight at 16°C And heat shock transformed into Escherichia coli TG1 competent cells, the clones identified as positive by colony PCR were enriched, and a small amount of plasmid was extracted by alkaline lysis method to obtain the cleavage vector.

[0043] Select 20 colonies of Escherichia coli TG1 containing the lysis vector, inoculate them in 5 mL of LB containing 50 μg / mL ampicillin, culture overnight at 37°C with shaking (220 r / min), then transfer 1-2 mL to 50 mL containing 50 μg / mL Ampicillin in LB, shake culture at 37°C to OD 600 Up to about 0.4. At this time, the temperature was raised rapidly to 42° C. for culture to induce the expression of...

Embodiment 3

[0044] Embodiment 3 Construction of the recombinant Haemophilus parasuis containing lysis vector pBV-Eprom

[0045] 1. Construct a lysis vector containing the bacteriophage lysis gene E of the mutation shown in SEQ ID NO: 1;

[0046] (1) Synthesizing the sequence of the mutated phage lytic gene E shown in SEQ ID NO: 1;

[0047] (2) Design primers:

[0048] Lysis E-U: 5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3' (SEQ ID NO. 2) Lysis E-L: 5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3' (SEQ ID NO. 3)

[0049] Using specific primers Lysis E-U and Lysis E-L to specifically amplify the sequence of the mutated phage lytic gene E synthesized above. The PCR amplification reaction system is 50 μL, in which MgSO 4 2mM, 1μM each for upstream and downstream primers, dNTP200μM, 10x Taq buffer, Taq TM DNA polymerase 2U (TaKaRa), template 1 μL. The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 30s, 59°C for 30s, 72°C for 30s, and 72°C for 5 minutes. Obtain the amplifie...

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Abstract

The invention discloses a method for preparing a bacterial ghost vaccine of haemophilus parasuis as well as a product and application thereof. The method comprises the following steps of connecting a mutational bacteriophage splitting gene E Eprom (as shown in SEQ ID No:1) with pBV220 to obtain an efficient splitting plasmid vector pBV-Eprom; converting the pBV-Eprom into haemophilus parasuis, propagating at 37 DEG C, and inducing the Eprom gene to express at 42 DEG C, and collecting the product which is unexpressed finally to obtain haemophilus parasuis bacterial ghost, wherein the Eprom is obtained by carrying out mutation on a promoter region of the bacteriophage splitting gene E, and the temperature for culturing bacteria is changed to 37 DEG C from the existing 28 DEG C by the Eprom; moreover, the splitting efficiency is high, the initial induced concentration and the large-scale production capacity are high, and the culture-splitting efficiency of a fermentation tank is as high as 99.99995%. The bacterial ghost vaccine of the haemophilus parasuis disclosed by the invention has good safety and immune protective efficacy, can be used for stimulating a body to generate a high-titration antibody, and also can be used for providing good cross immune protection for attack of a virulent strain of the haemophilus parasuis with different serotypes.

Description

technical field [0001] The present invention relates to a method for preparing haemophilus parasuis ghost vaccine and its products and applications, in particular to a method for preparing haemophilus parasuis ghost vaccine by using mutated phage cleavage gene E and the method obtained The invention relates to Haemophilus parasuis shadow vaccine and its application in the preparation of medicines for preventing and treating Haemophilus parasuis infection, belonging to the field of genetic engineering vaccines. Background technique [0002] Bacterial ghost is an empty bacterial body without cytoplasm and nucleic acid. The PhiX174 phage E cleavage gene is expressed in bacteria. The protein encoded by the gene can form a transmembrane tunnel on the bacterial cell membrane and cell wall, and the bacteria will rupture under the action of osmotic pressure. The cytoplasm and nucleic acid components in the bacterial cell pass through this tunnel. Expelled, forming an empty bacteria...

Claims

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Application Information

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IPC IPC(8): A61K39/102A61P31/04C12N15/74C12R1/21
Inventor 王春来张艳禾李刚谢芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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