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Application of recombinant bacillus subtilis in preventing or treating mycoplasma hyopneumoniae infection

A technology of Bacillus subtilis, B.S-P97R1, applied in the field of biotechnology genetic engineering, can solve the problem of not preventing Mycoplasma hyopneumoniae infection, and achieve the effect of ensuring vaccine safety, strong stress resistance and convenient application

Active Publication Date: 2019-04-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, there is no nasal spray vaccine on the market to prevent Mycoplasma hyopneumoniae infection

Method used

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  • Application of recombinant bacillus subtilis in preventing or treating mycoplasma hyopneumoniae infection
  • Application of recombinant bacillus subtilis in preventing or treating mycoplasma hyopneumoniae infection
  • Application of recombinant bacillus subtilis in preventing or treating mycoplasma hyopneumoniae infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of recombinant Bacillus subtilis B.S-P97R1 and B.S-P46 expressing Mycoplasma hyopneumoniae P97R1 and P46 genes

[0041] 1. Design and synthesis of primers:

[0042] According to the P97R1 and P46 gene sequences of 168 strains of Mycoplasma hyopneumoniae published in GenBank (gifted by researcher Feng Zhixin of Jiangsu Academy of Agricultural Sciences Veterinary Research Institute) (NC_017509.1) (see SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing), Primers were designed with the following sequences:

[0043] P97R1-F: 5'-CATTACCTCAGCCGCCAGCAG-3',

[0044] P97R1-R: 5'-AAGCCATTGGGAAATAGTCT-3';

[0045] P46-F: 5'-AAATGAAAAAAATGCTTAG-3',

[0046] P46-R: 5'-TTAGGCATCAGGATTATCAACAT-3';

[0047] The above primers were synthesized by Nanjing GenScript Biotechnology Company.

[0048] 2. Template preparation:

[0049] Take 168 cultured Mycoplasma hyopneumoniae strains, and use the boiling method to roughly extract the whole genome. The specific o...

Embodiment 2

[0078] Example 2 Test of Recombinant Bacillus subtilis B.S-P97R1, B.S-P46 Nasal Spray Immunization on Mice

[0079] 1. Select 80 6-week-old SPF female mice (Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing City), and randomly divide them into 4 groups, 20 in each group. Group 1: blank control group (PBS group, same dose of 20 μL sterile PBS nasal drop mice); group 2: empty vector group (B.S group, same dose (20μL 5×10 8CFU B.S) nasal drop mice); group 3: B.S-P97R1 group (on the 0th day nasal drop immunization 20μL 5×10 8 CFU B.S-P97R1, second immunization with the same dose of bacteria on the 28th day); group 4: B.S-P46 group (20μL 5×10 nasal drop immunization on the 0th day 8 CFU B.S-P46, second immunization with the same dose of bacteria on the 28th day), and on the 0th day, 7th day, 14th day, and 28th day after immunization, 4 animals in each group were killed to collect serum and alveolar washing fluid, and on the 42nd day all They were sacrificed, and seru...

Embodiment 3

[0082] Example 3 Recombinant Bacillus subtilis B.S-P97R1, B.S-P46 nasal spray immunization test of 21-day-old piglets

[0083] 1. Select 6 21-day-old SPF piglets (Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences), and randomly divide them into 2 groups, 3 piglets in each group. The immune group (immunized and challenged) was first immunized on the 0th day (1mL 5× 10 9 CFU B.S-P97R1 mixed 1mL 5×10 9 CFU B.S-P46 nasal spray immunization), 28 days of secondary immunization (immunization with the same dose); the control group (not immunized but challenged) with 2 mL sterile PBS nasal spray control. Serum was collected 14 days, 28 days, and 42 days after immunization (aseptically collect blood from the anterior vena cava into an anticoagulant tube, let it stand overnight at 4°C, centrifuge at 2500r / min at 4°C for 10min, collect the supernatant as a test sample, and store it at -20 ℃ frozen for later use) and nasal swabs (take the two nostril swabs of pigs...

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Abstract

The invention discloses application of recombinant bacillus subtilis B.S-P97R1 and B.S-P46 in the preparation of a drug for preventing or treating mycoplasma hyopneumoniae infection (porcine asthma).The recombinant bacillus subtilis B.S-P97R1 and B.S-P46 are successfully constructed. Firstly the B.S-P97R1 and the B.S-P46 immunize a mouse through nasal spray, so that effective specific IgG and SigA can be induced to be generated in a mouse serum and pulmonary alveoli rinsing liquid. In order to further verify the protective effect, after a 21-day-old piglet is immunized through nasal spray, achallenge test is carried out for 42 days, the growth state of the pig immune to the recombinant bacillus subtilis B.S-P97R1 and B.S-P46 is good, the clinical symptoms and lung pathological change are light, the capacity of an intrapulmonary mycoplasma is low, and a certain challenge protection function is achieved. The recombinant bacillus subtilis is expected to be developed into a geneticallyengineered nasal spray vaccine for preventing the mycoplasma hyopneumoniae.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, in particular to the application of recombinant bacillus subtilis B.S-P97R1 and B.S-P46 in preventing or treating mycoplasma hyopneumoniae infection. Background technique [0002] Mycoplasma hyopneumonia, also known as swine asthma, is a contact chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae, which exists widely in all parts of the world. It can infect pigs of all ages. Infection of pigs can lead to reduced feed conversion rate, poor growth and development, and secondary viruses or bacteria (porcine reproductive and respiratory syndrome virus, porcine circovirus, Streptococcus suis, parasuis Haemobacter, etc.) infection, causing pig mortality increased, resulting in heavy economic losses. [0003] Currently, immunization through vaccines is the most important means of preventing this disease. The Mycoplasma hyopneumoniae vaccines on the market are main...

Claims

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Application Information

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IPC IPC(8): A61K39/07A61K39/39A61P11/00A61P31/04C12N15/75
CPCA61K39/07A61K39/39A61K2039/543A61K2039/552A61K2039/55594A61P11/00A61P31/04C12N15/75
Inventor 杨倩王永恒李昱辰王佳璐
Owner NANJING AGRICULTURAL UNIVERSITY
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