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Establishment and application of surface display C type clostridium perfringens alpha and beta toxin protein recombinant lactobacillus plantarum

A technology for Clostridium perfringens and Lactobacillus plantarum, applied in recombinant DNA technology, microorganism-based methods, medical preparations containing active ingredients, etc., can solve the problem of strong virulence, incomplete inactivation, and difficulty in To achieve the inactivation conditions and other issues, to achieve a good protective effect

Pending Publication Date: 2019-10-01
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional inactivated vaccines have shortcomings such as incomplete inactivation and strong virulence.
At present, toxoid vaccines have been widely used, but because the inactivating substance is formaldehyde, the loss of antigen is relatively large, and it is difficult to achieve a satisfactory inactivation condition

Method used

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  • Establishment and application of surface display C type clostridium perfringens alpha and beta toxin protein recombinant lactobacillus plantarum
  • Establishment and application of surface display C type clostridium perfringens alpha and beta toxin protein recombinant lactobacillus plantarum
  • Establishment and application of surface display C type clostridium perfringens alpha and beta toxin protein recombinant lactobacillus plantarum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 illustrates the construction method of recombinant Lactobacillus plantarum for surface display of Clostridium perfringens type C α, β toxin protein:

[0050] 1. Amplification of the target gene:

[0051] According to the three toxin gene sequences of Clostridium perfringens type C on GenBank (M24904.1, KP064405.1, L77965.1), primers were designed in combination with the actual conditions of the expression sequence and the vector.

[0052] α-F: 5'-GGC TCTAGA TGGGATGGAAAGATTGATGGAACAGG-3'

[0053] α-R: 5'-GGC AAGCTT TTTTATATTATAAGTTGAATTTCCTGAAATCCACTC-3'

[0054] β1-F: 5'-GGC TCTAGA ATGAAGAAAAAATTTATTTCATTA-3'

[0055] β1-R: 5'-CGG GGTACC CTAAATAGCTGTTACTTTGTGAGT-3'

[0056] β2-F: 5'-GGC TCTAGA ATGAAAAAAATTTTTCAAAA-3'

[0057] β2-R: 5'-CGG GGTACC CTATGCACAATACCCTTCAC-3'

[0058] The primers were synthesized by Harbin Xinhai Genetic Testing Co., Ltd. XbaI, HindIII and KpnI restriction endonuclease sites were introduced at the 5' end of the primers ...

Embodiment 2

[0078] Embodiment 2 illustrates recombinant plant lactobacillus identification:

[0079] 1. Determination of the expressed target protein concentration by BCA method:

[0080] The three strains of recombinant Lactobacillus plantarum were induced to express and subjected to ultrasonic crushing. For the specific operation steps, refer to the instructions of Beyontian BCA Protein Concentration Detection Kit, and the concentration of protein expressed by recombinant Lactobacillus plantarum in each group.

[0081] Table 2 BCA detection protein concentration

[0082] Recombinant Lactobacillus plantarum group Protein concentration (mg / ml) pSIP409-pgsA'-α 0.11148 pSIP409-pgsA'-β1 0.21048 pSIP409-pgsA'-β2 0.21106 pSIP409-pgsA' 0.42870

[0083] 2. Western-blot detection:

[0084] In order to infer whether the expressed protein is expressed on the surface of the bacteria, two methods were used to process the protein sample, ultrasonic disruption an...

Embodiment 3

[0088] Example 3 Evaluation of the immune effect of recombinant Lactobacillus plantarum expressing Clostridium perfringens α, β toxin protein:

[0089] 1. Animal immunization groups

[0090] Take 150 5-week-old female BALB / c mice, divide them into 3 groups on average, raise them separately, and mark them well. Centrifuge the recombinant Lactobacillus plantarum that has been constructed and induced overnight at 5000r / min for 6min, and wash the bacterial pellet with sterilized PBS. After repeated washing three times, calculate the number of viable bacteria, and the number of viable bacteria for each immunization is 1× 10 9 cfu. The first immunization was carried out on the first day, the third day, and the fifth day, and the booster immunization was carried out on the 20th day, the 22nd day, and the 24th day. Before oral immunization, feeding was fasted for 4 to 6 hours, and sufficient drinking water was ensured during the period, and blood was collected every week. Feces and...

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Abstract

The invention relates to surface display C type clostridium perfringens alpha and beta toxin protein recombinant lactobacillus plantarum. The C type clostridium perfringens alpha toxin gene sequence,beta1 toxin gene sequence and beta2 toxin gene sequence are inserted in a genome of lactobacillus plantarum. The invention further relates to an establishment method and application of the recombinantlactobacillus plantarum. Through a PCR method, alpha, beta1 and beta2 toxin genes are successfully cloned on the whole genome of C type clostridium perfringens (CVCC 1158), a corresponding recombinant plasmid is established and electrically transferred into lactobacillus plantarum NC8, three strains of recombinant lactobacillus plantarum are obtained after induction expression, the oral mode is selected for immunizing a rat, the immune effect of recombinant bacteria is evaluated, the result shows that when the rat is immunized by orally taking the recombinant lactobacillus plantarum, the ratbody can be stimulated to generate humoral immunity and can also be stimulated to generate cellular immunity, and a good protection effect on an animal body is realized.

Description

technical field [0001] The invention belongs to the field of animal molecular biology, and particularly relates to the construction and application of a recombinant plant lactobacillus displaying C-type α and β toxin proteins of Clostridium perfringens on the surface. Background technique [0002] Clostridium perfringen (Clostridium perfringen), also known as Clostridium welchii, is an anaerobic bacillus that can produce pathogenic exotoxins. There are many types of exotoxins produced by Clostridium perfringen, among which α, β , ε, and ι are the main lethal toxins. According to the different types of lethal toxins, Clostridium perfringens can be divided into five types: A, B, C, D, and E. α-toxin and β-toxin are C-type gas-producing The major virulence toxin of Clostridium perfringens. The α-toxin is an important pathogenic factor during Clostridium perfringens infection. Among many bacterial toxins, this toxin is the first bacterial protein that not only has enzymatic act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/08A61P31/04C12R1/25
CPCA61K39/08A61K2039/542A61P31/04C07K14/33C12N15/746
Inventor 王欣李超倪宏波
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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