High-level expression of tetanus toxin receptor binding domain Hc in Escherichia coli and application

Abstract

The invention relates to a method for a tetanus toxin receptor binding domain Hc to be subjected to high-level soluble expression in Escherichia coli through nucleotide sequence optimization. According to the sequencing result of a domestic C.Tetani virulent strain CMCC64008, the tetanus toxin receptor binding domain Hc sequence is analyzed and optimized, the optimized sequence is SEQ ID No.1 and the coded protein sequence is SEQ ID No.2. The synthesized Hc gene is linked into an expression vector pET32a(+) after undergoing double enzyme digestion, the recombinant Hc is subjected to high soluble expression in Escherichia coli and the target protein accounts for about 46% of the total protein in the supernatant undergoing bacteriociasis. After QFF column purification, phenyl hydrophobic column purification and SP column purification, the purity of the target protein can be more than 95% and the yield thereof is more than 300mg / L. The recombinant protein prepared by the method of the invention has good immunogenicity, can induce the mice to produce high-titre protective antibodies and can resist attack of high-dose lethal toxins. The method has extensive application prospect in large-scale high-level preparation of the tetanus toxin recombinant subunit vaccine Hc.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tetanus toxin receptor binding region Hc gene and its coded protein and its application as a subunit vaccine in tetanus prevention. technical background: [0002] Tetanus is a disease that seriously endangers people's lives and health. It is an acute and specific disease caused by Gram-positive, anaerobic Clostridium-tetani bacilli invading human wounds, growing and multiplying, and producing tetanus toxin. Sexual infection, tetanus toxin invasion of the nervous system can lead to generalized muscle spasm, and then systemic failure or suffocation to death. All open injuries have the possibility of tetanus. The toxicity of tetanus toxin is very strong, second only to botulinum toxin, and about 100ng of tetanus toxin can cause death. Tetanus is very serious in the vast poor and backward third world countries and regions. It is estimated that about 1,00...

AI Technical Summary

Problems solved by technology

Traditional toxoid vaccines are refined from tetanus toxin through formaldehyde detoxification and aluminum adjuvant adsorption. Although the immune effect is good, there are still some problems: certain side effects occur after inoculation of toxoid; Bacteria can form spores, which are highly toxic, and there is a certain risk in the production of vaccines; toxoids treated with formaldehyde are likely to cause pollution; toxoids after chemical treatment may undergo toxicity reversal; and the important thing is that the immunity period is short. Tetanus immunity declines, children need booster immunization every 4 to 6 years, adults need booster immunization every 10 years

[0004] It is of great significance to use E. coli to express the subunit vaccine against tetanus toxin. The process is simple, and the recombinant Hc protein does not need to worry about being contaminated by the complete toxin, and its safety is relatively high. However, the currently expressed tetanus Hc fragment is generally expressed Low amount, easy to form inclusion bodies, low immune titer and other disadvantages, this is because the AT content in the Hc sequence is rich and there are a large number of rare codons in Escherichia coli, which limits its expression to a certain extent

Many researchers at home and abroad have improved the expression of the tetanus Hc segment, and the expression level has been increased to a certain extent, but it still cannot meet the needs of mass production, and the prepared Hc immunogenicity is also lower than that of traditional toxoid vaccines. lower

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