Clostridium welchii disease resistant transgenic plant vaccine and preparation method thereof

Abstract

The invention discloses a clostridium welchii disease resistant transgenic plant vaccine and a preparation method thereof. At present, the protective ratio on immunization of clostridium welchii disease resistant vaccines is lower. The preparation method of the clostridium welchii disease resistant transgenic plant vaccine comprises the steps of: (1) with a plasmid or disease sample containing coded clostridium welchii toxin as a template, carrying out PCR (Polymerase Chain Reaction) or RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification to obtain a clostridium welchii toxin gene, carrying out enzyme cutting on the obtained clostridium welchii toxin gene or the Ti or Ri plasmid respectively, recycling a target gene and a plant expression vector segment, connecting with and converting escherichia coli, carrying out enzyme cutting, PCR and sequencing identification on the recombinant Ti or Ri plasmid, converting agrobacterium tume faciens or agrobacterium rhizogene with the recombinant plasmid, and screening and identifying positive recombinant agrobacterium; and (2) carrying out vernalization, germination and splicing on plant seeds to obtain explants, infecting the explants respectively with the recombinant agrobacterium in the step (1), carrying out screening, subculturing, rooting and transplanting on the infected explants to obtain transformed plants, and screening and identifying positive transgenic plants.

Description

Technical field: [0001] The invention relates to a transgenic plant vaccine against Clostridium welchii disease and a preparation method thereof. Background technique: [0002] Clostridium welchii (Clostridium welchii), also known as Clostridium perfringens (Clostridium perfringens) is one of the normal flora in the intestinal tract of animals, and it is also the original culprit that causes Clostridium welchii disease. The pathogenesis is that Clostridium welchii multiplies in large numbers in the intestines due to sudden changes in the feeding environment and feed, and the toxins produced are absorbed through the digestive tract, leading to enterotoxemia, necrotic enteritis, soft kidney disease in sheep, and red piglets. Dysentery, lamb dysentery, rabbit hemorrhagic diarrhea, traumatic gas gangrene, animal sudden death syndrome, etc. According to the different exotoxins secreted by the bacteria, Clostridium welchii can be divided into five serotypes, namely A, B, C, D, an...

AI Technical Summary

Problems solved by technology

[0009] At present, formaldehyde is still the most commonly used chemical substance for exotoxin inactivation. Its inactivation effect is mainly related to the concentration, temperature, action time and pH of formaldehyde. High concentration formaldehyde and high temperature are used for inactivation. Although the inactivation time can be shortened, the effect on the antigen The loss of formaldehyde solution is also very large. Formaldehyde solution has a reducing effect and can inhibit protein activity. It also involves the issue of retaining the antigenicity of the toxin and eliminating its toxicity to the greatest extent. Due to many factors affecting inactivation, it is difficult to achieve an ideal inactivation. condition

The formaldehyde-detoxified Clostridium welchii α-toxin toxoid has a protective effect after immunizing sheep, but the toxoid may also contain other antigenic components of Clostridium welchii, which requires multiple intraperitoneal injections for immunization, and the inactivation of formaldehyde is not complete Clostridium welchii causes environmental pollution. The biggest problem is that the time required for toxoid-induced antibody production often exceeds the time for gas gangrene symptoms to appear

At the same time, inactivated vaccines are not easy to stimulate the body to produce local mucosal immunity, and the ability to mediate cellular immunity is weak, which reduces the immune efficacy of inactivated vaccines. Moreover, the manufacturing process is cumbersome, and it must be inoculated through non-oral routes to induce the body to produce circulating antibodies. Difficulty producing secreted local antibodies and therefore not blocking absorption of toxins from mucous membranes into the body

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