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Recombinant lactic acid bacteria of pig interleukin 10 and application thereof

A technology of interleukin and recombinant lactic acid bacteria, applied in application, bacteria, recombinant DNA technology and other directions, can solve the problems of decreased immunity and anti-stress ability of pigs, and achieve the goal of promoting sustainable and healthy development, ensuring healthy growth, and enhancing disease resistance. force effect

Inactive Publication Date: 2011-04-13
GUANGDONG TIANHAO BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problem of pig immunity and anti-stress ability decline under high-density and intensive breeding conditions, and to improve the ability of pigs to produce specific antibodies against pathogenic microorganisms, and to provide oral, non-injection Recombinant lactic acid bacteria capable of stably expressing porcine interleukin-10 with immune regulation function

Method used

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  • Recombinant lactic acid bacteria of pig interleukin 10 and application thereof
  • Recombinant lactic acid bacteria of pig interleukin 10 and application thereof
  • Recombinant lactic acid bacteria of pig interleukin 10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of porcine interleukin 10 (poIL-10) gene

[0040] Take pig spleen, make spleen cell suspension, use Trizol to extract splenocyte RNA, take 1μl and carry out 0.8% agarose gel electrophoresis detection, such as figure 1 shown.

[0041] 1) Amplification of poIL-10 gene

[0042] Primers were designed according to the poIL-10 gene sequence registered in GenBank, upstream primer: 5′TTT GGTACC ATGCCCAGCTCAG, downstream primer: CCC AAGCTT TCAGTTCTTCCTCATC, containing Kpn I and HindIII restriction sites respectively, was sent to Shanghai Sangong for synthesis. Using splenocyte RNA as a template, the poIL-10 gene was amplified by RT-PCR.

[0043] reaction system:

[0044] Upstream primer (P1, 10 μmol / L) 1.0 μL

[0045] Downstream primer (P2, 10μmol / L) 1.0μL

[0046] dNTP (2.5mmol / L) 4.0μL

[0047] 10 × Buffer 5.0μL

[0048] Transcriptase-TapDNA Polymerase 1.0 μL

[0049] Template RNA 1.0 μL

[0050] wxya 2 O 37.0 μL

[0051] ...

Embodiment 2

[0075] Example 2 Construction of recombinant expression vector pW25et-poIL-10

[0076] 1) Preparation of plasmid pW425et

[0077] Escherichia coli liquid containing pW425et was prepared, and the plasmid was extracted according to the instructions of the plasmid DNA extraction kit, and 2 μL was taken for 0.8% agarose gel electrophoresis detection. The results showed that the plasmid pW425et was successfully obtained, as Image 6 shown.

[0078] 2) Recovery, connection and transformation of the target gene

[0079] Recovery of the target gene:

[0080] The pMD8T-poIL-10 and pW425et were double-digested with Kpn I and HindIII restriction endonucleases respectively to obtain the large fragment of the poIL-10 gene and pW425et; the double-digestion system was as follows:

[0081] 10×L Buffer 2.0μL

[0082] Recombinant plasmid (or vector) 16.0μL

[0083] HindIII 1.0 μL

[0084] KpnI 1.0 μL

[0085]

[0086] Total V 20.0μL

[0087] Reaction conditi...

Embodiment 3

[0108] Example 3 Preparation of recombinant lactic acid bacteria expressing porcine interleukin 10

[0109] 1) Preparation and transformation of Lactobacillus competent cells

[0110] Incubate Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus reuteri or Lactobacillus fermentum frozen at -70°C until thawed and spread on MRS plate (containing thymidylic acid 100 μg / mL), anaerobic static culture at 37°C overnight. The next day, pick colonies and inoculate them in fresh 10mL MRS liquid medium (containing 1% glycine), and anaerobic static culture at 37°C until the cells OD 600 The value is 0.6-0.8. Take the bacterial cell culture solution, inoculate it in fresh MRS liquid medium (containing 1% glycine) according to the dose of 1%-2%, and culture it anaerobically at 37°C until the cell OD 600 When the value is 0.2-0.3, collect and reserve;

[0111] Place the bacterial culture collected above...

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Abstract

The invention relates to recombinant lactic acid bacteria of pig interleukin 10. Ribose nucleic acid (RNA) in pig spleen cells is used as a template; poIL-10 genes are obtained by reverse transcription-polymerase chain reaction (RT-PCR) technology; the poIL-10 genes are transformed into escherichia coli after being connected with a pMD18T vector of a cloning vector; and the poIL-10 genes are inserted into an escherichia coli-lactic acid bacterium shuttle expression plasmid pW425et to build a recombinant plasmid pW425et-poIL-10 and electrically transform the recombinant plasmid pW425et-poIL-10into the lactic acid bacteria to obtain the recombinant lactic acid bacteria capable of stably expressing the pig interleukin 10. The recombinant lactic acid bacteria can be planted in intestinal canals through oral administration, stably express the pig interleukin 10, effectively regulate immune functions of organisms, stimulate proliferation of cells B, improve the levels of IgG, IgM and IgA of serum, improve the capability of the organisms generating specific antibodies for pathogenic microorganisms, ensure healthy growth of pigs and promote sustainable and healthy development of animal husbandry.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a porcine interleukin-10 recombinant lactic acid bacterium and an application thereof. Background technique [0002] Animal husbandry is an important part of the modern agricultural industrial system, an advantageous industry in the strategic adjustment of my country's agricultural and rural economic structure, and also the main way to transform the rural economic growth mode and one of the important contents of the new rural construction. my country is a big pig producing country and a big pork consuming country. Pork is the main source of meat food for our people, accounting for more than 60% of daily meat consumption. Although the intensive and high-density breeding mode solves the problem of consumption of pork products due to the increase in population density, due to the highly intensive farming conditions, the immunity and anti-stress ability of pigs are reduced, and the chanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/24C12N15/74A23K1/16C12R1/225C12R1/25A23K10/18A23K50/30
Inventor 彭永鹤王春凤杨桂连杨文涛郝凤奇石海宁
Owner GUANGDONG TIANHAO BIOLOGICAL ENG
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