Method for preparing tetanus toxin recombinant antigen and its use

A tetanus toxin and tetanus technology, applied in the field of genetic engineering, can solve the problems of low recovery rate, high toxicity, complicated C fragment steps, etc., and achieve the effects of good immunogenicity, prevention of hydrolysis, and easy reproduction.

Inactive Publication Date: 2010-04-28
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many disadvantages by directly extracting and purifying the C fragment from tetanus toxin: Clostridium tetani can form spores, and its toxicity is high, and there is a certain risk in the cultivation and isolation process; The procedure of C fragment is complicated and the recovery rate is not high

Method used

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  • Method for preparing tetanus toxin recombinant antigen and its use
  • Method for preparing tetanus toxin recombinant antigen and its use
  • Method for preparing tetanus toxin recombinant antigen and its use

Examples

Experimental program
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Embodiment 1

[0041] Embodiment 1, the amplification of tetanus toxin antigenic protein gene

[0042] (1) Preparation of amplification template

[0043] The toxin-producing strain CMCC64008 of Clostridium tetani was selected as the amplified strain. Inoculate the bacteria in bract meat culture medium, culture anaerobically at 35°C for 2 to 3 days, centrifuge at 4000rpm for 20 minutes, discard the supernatant, suspend the bacterial pellet in distilled water, freeze and thaw repeatedly in liquid nitrogen and room temperature 3 times, boil in boiling water for 5- 10 minutes, centrifuged at 10000rpm for 30 minutes, and the supernatant of the bacterial lysate was taken as the amplification template.

[0044] (2) Primer design

[0045] According to the tetanus toxin gene sequence in GenBank, primers with BamH I and Hind III restriction sites (indicated in italics) at both ends of the gene were designed, as shown in Table 1 below.

[0046] Table 1: Primer sequences

[0047]

[0048] (3) PCR...

Embodiment 2

[0050] Cloning and sequencing of embodiment 2, PCR product

[0051] (1) Purification of PCR products (glass milk purification)

[0052]After the PCR product obtained in the previous steps was separated by 1% agarose (Sigma company) gel electrophoresis, the target band was cut out under the irradiation of a long-wave ultraviolet lamp, and placed in a 1.5ml Eppendorf tube. Add 3 times the volume of sol solution (100 μl volume of gel to add 300 μl sol solution), 65 ° C for 5 minutes, during which the Eppendorf tube was shaken several times to completely dissolve the gel. Add 10 μl of glass milk (product of Boda Company) (mix the glass milk thoroughly before use), invert and mix, place in ice bath for 10 minutes, and mix every 2-3 minutes. Centrifuge at 12,000 rpm for 30 seconds, and discard the supernatant. Add 250 μl of rinse solution, blow the rinse solution with a pipette, gently suspend and mix the glass milk, centrifuge at 12,000 rpm for 30 seconds, and discard the superna...

Embodiment 3

[0069] Embodiment 3, construction of recombinant expression plasmid

[0070] The target gene on both sides of the recombinant cloned plasmid pGEM-T / ttc obtained in Example 2 has restriction endonuclease BamH I, Hind III recognition sites, and it is digested with these two enzymes to obtain the target gene. The expression vector pET-22b(+) (see Figure 5 ) connection, its construction diagram is shown in Figure 6 , transform the competent cells of Escherichia coli BL21 (DE3), extract the plasmids, and obtain positive clones through enzyme digestion identification. Agarose electrophoresis identification, there is a 1.3Kb fragment, which is the same size as ttc, see Figure 7 . The expressed plasmid is pET-22b(+) / ttc, and the reading frame analysis is shown in Table 3.

[0071] Table 3: Analysis of pET-22b(+) / ttc reading frame

[0072]

[0073]

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Abstract

The present invention relates to the recombinant vector of tetanus toxin antigen protein gene, the vector transformed host, tetanus toxin antigen produced with the transformant, and the application ofthe antigen protein in tetanus vaccine and conjugated vaccine protein vector. The recombinant plasmid vector of the present invention contains tetanus toxin antigen protein gene and is constituted viaconnecting tetanus toxin antigen protein gene and prokaryotic expression vector pET-22b(+). The transformant is obtained via transforming colibucillus with the recombinant plasmid. The expression sys tem of the present invention has high efficiency expression of tetanus toxin antigen protein with expression amount of 15 %, and the purified expression protein has purity up to 96.5%. The tetanus toxin antigen of the present invention high immunogenicity, and may be used in preparing tetanus vaccine and the vector protein of conjugated vaccine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector containing a tetanus toxin antigen gene, a host transformed with the vector and a transformant used to produce the tetanus toxin antigen, and the antigen in a tetanus vaccine and a combination vaccine protein carrier in the application. technical background [0002] Tetanus is caused by the infection of Clostridium tetani (Clostridium tetani). Under anaerobic conditions, bacteria produce a large amount of toxin-tetanus neurotoxin (abbreviated as tetanus toxin). Sexual muscle tonic spasm, severe cases finally died of suffocation and systemic failure. It is generally estimated that about 1 million people die from tetanus each year, about 80% of whom are newborns. Due to its sinister symptoms, high morbidity and mortality, tetanus has been highly valued by the medical profession. The most effective way to control tetanus is to vaccinat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N15/00C12N15/31A61K39/08C12P21/02A61P31/04
Inventor 雷殿良张庶民谭亚军
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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