Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea

A technology of recombinant protein and expression system, applied in the field of biomedicine, can solve the problem of no vaccine, etc., and achieve the effects of good specificity, good effect and good protection effect

Active Publication Date: 2021-11-23
潍坊峡山维泰生物科技有限公司
View PDF11 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After research, it is found that one of the main causes of diarrhea in piglets is infectious bacterial diarrhea, and piglet colibacillosis is a type of disease caused by pathogenic Escherichia coli Infectious diseases, among which enterotoxin-producing Escherichia coli (enterotoxingenic Escherichia coli, ETEC) is an important pathogen causing diarrhea in piglets; in addition, red scour in piglets caused by Clostridium welchii is also an important infectious bacterial diarrhea in piglets, but there is currently no A vaccine that can simultaneously prevent and control multiple pathogenic bacteria that cause diarrhea in piglets

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea
  • Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea
  • Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Find the amino acid sequence (BAA25725.1) and LTIT (BAA25726.1), LTIAA (WP_095374651.1) and LTIAB (WP_095389218.1), LTIAB (WP_095389218.1), LTIAB (WP_095389218.1), LTIAB (WP_095389218.1), LTIAB (WP_095389218.1), LTIAB (ALO79807.1) ) And ltincb (alo79813.1), heat-resistant colorectoxin ST A (CAD87828.1) and ST B (CAD87835.1) and α toxins (AQN80672.1) and beta toxins (Aji77135.1) Relevant information, antigenic analysis is performed by DNASTAR.

[0066] Depending on the amino acid sequence of α toxins LTIA, LTIB, LTICA, LTICB, ST A, ST B, LTICA, LTICB, ST A, ST B, LTICA, LTICB, ST A, ST B, LTLCA, and the amino acid sequence of beta toxins were predicted to predict the antigenicity of antigenicity.

[0067] LTIA's No. 74-113-amino acid residue of LTIAA, 1,1263 amino acid residues of LTIAA, LTIAA, 74th to 263 amino acid residues, LTIABs, 1,263 amino acid residues, LTIAA, 1,163 amino acid residues, LTIAA At the 20th to 50th amino acid residues of LTICA, the 64th to 90 amino a...

Embodiment 2

[0105] LTⅠ-LTⅡ-ST-Cp activity verification and purification of recombinant proteins

[0106] 1, Protein Purification

[0107] 1) The LTⅠ-LTⅡ-ST-Cp recombinant E.coli BL21 (DE3) bacteria, before 100μL inoculated in 5ml LB liquid added thousandth of kanamycin penicillin, 37 ℃, 160rpm shaking culture 3h.

[0108] 2) the bacteria in step 1 5ml access 1L sterilized LB added thousandth of kanamycin penicillin, 37 ℃, 160rpm, shaking culture 2h, then added of IPTG, to a final concentration of 1mmol / L, 16 ℃ shaking 160rpm, inducing 24h.

[0109] 3) The bacterial solution from step 2, 8000rpm, 4 ℃ centrifuged 10min, close bacteria, sonicated for PBS, the ice bath was sonicated on ice for 10min bacteria, ultrasonic distance 3s 3s, total 30min, 8000 rpm for ultrasonic completed, centrifuged 4 ℃ 10min, supernatant was discarded, received precipitation. Washed 3 times with PBS, and centrifugation conditions as before.

[0110] 4) Take the resulting precipitate was further weighed wet weight, ...

Embodiment 3

[0152] Recombinant protein LTI-LTITI-ST-CP on the immunocant protection of piglets

[0153] 1, experimental strain and animal

[0154] The new born long white pig, purchased from the Yangchun pig farm (the pig farm did not have a pig diarrhea, and if the pig is not vaccinated after birth), purchase 50 piglets, raised in standard pig houses, professionals according to standards Feeding, each pig is individually crossed, and any antibiotics are not added in the feed.

[0155] 2, the preparation of vaccines

[0156] The recombinant protein was diluted to 1 μg / μL, and the aluminum hydroxide sol adjuvant was slowly added to the recombinant protein (1G adjuvant per piglet), and was slowly shaken at room temperature for 30 min at room temperature.

3,

[0157] 3.1 vaccination and immunization

[0158] 1) Experiment group

[0159] 40 head pigs were randomly divided into 4 groups, and 10 per group was immunized according to the following immunization procedures, and the immunization cycl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to view more

Abstract

The invention provides a construction method, an expression system and application of a multi-union fusion recombinant protein capable of preventing piglet diarrhea, and relates to the technical field of biological medicines. According to the construction method, the expression system and application of the multi-union fusion recombinant protein capable of preventing piglet diarrhea, heat-resistant enterotoxin and heat-labile enterotoxin genes of ETEC and clostridium welchii alpha toxin and beta 1 toxin genes are cloned, functional areas of part of the genes are connected in series by using an SOE-PCR method, an LTI-LTII-ST-Cp recombinant sequence is amplified, an expression vector is successfully constructed, and the recombinant protein LTI-LTII-ST-Cp is successfully expressed. Each segment of the recombinant protein LTI-LTII-ST-Cp is good in specificity, the recombinant protein is non-toxic after immunizing mice, the antibody titer is relatively high, and the recombinant protein has a very good protection effect on diarrhea. According to the construction method, the expression system and application of the multi-union fusion recombinant protein capable of preventing the piglet diarrhea, the protection rate of the prepared vaccine on piglet red scour and piglet yellow scour can reach 80% or above, the effect is good, and the vaccine has very high protection capacity.

Description

Technical field [0001] The present invention belongs to the field of biotechnology medicine, particularly relates to a multi-preventing diarrhea in piglets fusion protein construct recombinant expression systems and methods of application. Background technique [0002] Diarrhea is a typical high intensive pig disease under production conditions. The disease is one of the most serious pig disease group, is caused by an important cause of death of piglets. According to the survey, piglets due to diarrhea deaths can be accounted for 39.8% of the total number of piglets died. Therefore, how to take effective measures to prevent and control diarrhea and improve the survival rate of piglets has become an important topic in the pork industry production. [0003] The study found that one of the main causes of diarrhea caused by the occurrence of infectious bacterial diarrhea, piglet E. coli disease is a type of infectious diseases caused by pathogenic E. coli, which enterotoxigenic E. co...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/31C12N15/70A61K39/02A61K39/07A61P1/12A61P31/04
CPCC07K14/245C07K14/33C12N15/70A61K39/025A61K39/07A61P31/04A61P1/12C07K2319/00A61K2039/70Y02A50/30
Inventor 王海波刁鹏飞谭双邓红娟李洪凌马学东曹旗任洪林
Owner 潍坊峡山维泰生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap