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Avirulent C-type clostridium botulinum gene engineering subunit vaccine and production method thereof

A subunit vaccine, Clostridium botulinum technology, applied in the direction of vaccines, DNA / RNA vaccination, antibacterial drugs, etc., can solve the problem of difficult to improve the refolding rate, to maintain effectiveness, reduce preparation time and production costs , good immune effect

Active Publication Date: 2019-08-02
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and the renaturation rate is often difficult to increase. This is the main factor limiting its application, and the use of soluble expression can overcome this problem very well. a question

Method used

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  • Avirulent C-type clostridium botulinum gene engineering subunit vaccine and production method thereof
  • Avirulent C-type clostridium botulinum gene engineering subunit vaccine and production method thereof
  • Avirulent C-type clostridium botulinum gene engineering subunit vaccine and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] ——Construction, expression and identification of Escherichia coli BL / BoM strain

[0077] 1. Coding rMBP-BoH C synthesis of genes

[0078] This application is based on the avirulence region H of maltoglycoprotein (MBP) and Clostridium botulinum toxin C (BoH C ) of the natural coding gene sequence (sequence 1), which was concatenated after codon optimization, and the coding sequence of the 6×His tag used was added at the 3' end of the concatenated gene. The gene sequence was synthesized by chemical synthesis method, GMBPBoH C . The specific nucleic acid sequence is shown in SEQ ID No.2, and the amino acid sequence is shown in SEQ ID No.3.

[0079] Sequence 1 (Clostridium botulinum toxin heavy chain amino acid sequence)

[0080]

[0081]

[0082] Sequence 2 (coding recombinant clostridium botulinum toxin (rMBP-BoH C ) gene nucleotide sequence)

[0083]

[0084]

[0085] Sequence 3 (recombinant Clostridium botulinum toxin (rMBP-BoH C ) amino acid sequen...

Embodiment 2

[0100] ——rMBP-BoH C expression and identification of

[0101] 1. rMBP-BoH C The expression of genetically engineered bacteria Escherichia coli BL / BoM strain was inoculated in 3 mL of LB liquid medium containing kanamycin, placed in 37 ° C shaking culture, when OD 600 When the temperature is 0.6-0.8, add IPTG solution with a final concentration of 0.5mM and place at 37°C and 15°C for induction and culture for 4h and 16h, respectively. After the bacterial culture was completed, the bacterial cells were collected by centrifugation, and the bacterial cells were resuspended at a ratio of 10 mL of lysate [0.02 mol / L Tris buffer (pH 7.2), 0.3 mol / L NaCl] per gram of bacterial body weight, and placed in an ice-water bath. The bacteria were disrupted by ultrasonic for 30 minutes, the crushing conditions were: working for 9s, resting for 9s, and the ultrasonic power was 400W. The crushed bacterial liquid was centrifuged at 12000r / min for 10min at 4°C, and the supernatant was collecte...

Embodiment 3

[0104] ——rMBP-BoH C purification of

[0105] Inoculate the genetically engineered Escherichia coli BL / BoM strain in 1L of LB liquid medium containing kanamycin for fermentation culture, shake culture at 37°C until OD 600 When the temperature is 0.6-0.8, adjust the temperature to 15°C, add IPTG solution with a final concentration of 0.5mM to induce culture for 4h. After the bacterial culture was completed, the bacterial cells were collected by centrifugation, and the bacterial cells were collected by centrifugation at 5000r / min for 5min, and 10ml of lysate (pH value 7.2 0.02mol / L Tris buffer solution, 0.3mol / L NaCl) was added per gram of bacterial cell wet weight. Proportionally resuspend the bacteria, and break the bacteria three times with a low-temperature high-pressure homogenizer at a pressure of 800 bar at 4°C. The lysate was centrifuged at 10,000 r / min at 4°C for 30 min, and the supernatant was collected. According to the instruction manual of the Ni-IDA affinity chrom...

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Abstract

The invention relates to an avirulent C-type clostridium botulinum gene engineering subunit vaccine and a production method thereof. The prepared avirulent C-type clostridium botulinum subunit vaccineis produced by adopting recombinant protein (rMBP-BoHC), which is subjected to codon optimization and contains a maltose-binding protein (MBP) tag, of an avirulent area HC (BoHC) of a clostridium botulinum toxin (BoNTs) heavy chain, so that not only is the safety of the vaccine guaranteed furthest, but also the effectiveness of the vaccine is maintained. Meanwhile, an antigen of the vaccine can be expressed in a soluble mode, so that the avirulent C-type clostridium botulinum gene engineering subunit vaccine has the advantages of being simple in preparation technology, low in immunizing dose,good in immune efficacy and the like, and is an ideal candidate vaccine for upgrading and updating current C-type clostridium botulinum vaccines of the nation.

Description

technical field [0001] The invention relates to a non-toxic Clostridium botulinum type C genetic engineering subunit vaccine and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium botulinum (Clostridium botulinum) poisoning is a very serious fatal disease caused by Clostridium botulinum Neurotoxins (BoNTs), which not only seriously endangers human health, but also causes significant economic losses to animal husbandry. . Currently, there are seven known serotypes of Clostridium botulinum, A-G. Among them, Clostridium botulinum type C is the most harmful to animal husbandry. In many countries and regions, including Brazil, Europe and North America, there are reports of livestock botulism events, and the mortality rate can be as high as 99.92% and above (Gil L A F, Cunha C, Moreira G, et al. Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Type...

Claims

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Application Information

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IPC IPC(8): A61K39/08A61P31/04
CPCA61K39/08A61P31/04A61K2039/523A61K2039/53Y02A50/30
Inventor 陈小云薛麒刘莹李旭妮杜吉革李启红朱真张莹辉印春生姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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