Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof

A technology of immune regulation and lactic acid bacteria, applied in the biological field, can solve problems such as limited production, poor control of immune procedures and methods of use, rejuvenation of coccidia, etc., and achieves simple preparation methods and production processes, and is convenient for large-scale production and application. The effect of broad application prospects

Inactive Publication Date: 2013-09-04
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these vaccines all have their own shortcomings: (1) the immunization procedure and the method of use are not easy to control; (2) the production volume is limited, and it is difficult to meet the needs of the modern chicken industry; (3) the coccidia may rejuvenate and return to strength, etc., Therefore, it is difficult to apply widely

Method used

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  • Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof
  • Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof
  • Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 SO7 Gene acquisition

[0027] (1) Design and synthesis of primers

[0028] According to Eimeria tenella published in GenBank SO7 Gene sequence (GI: 9324) uses Primer5.0 software to design primers, and the upstream and downstream primers are designed separately Nco I. Kpn Ⅰ Restriction site, primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The primer sequences are as follows:

[0029] SO7 Upstream: 5-AT CCATGG CAGACCTCTTCAGCGG-3

[0030] SO7 Downstream: 5-TA GGTACC TGGCCGTTGTGGTGTTGA-3

[0031] (2) SO7 PCR amplification of genes

[0032] The total RNA of the Eimeria tenella Beijing strain was extracted by the conventional Trizol method, reverse-transcribed into cDNA, and the cDNA was used as a template to amplify the target gene with a size of about 651bp using the above primers, as shown in figure 1 shown. Its base sequence is shown in the sequence table SEQ ID NO.1.

[0033]

[0034]

[0035] PCR ...

Embodiment 2

[0038] Example 2 ChIL-2 Gene acquisition

[0039] Published in Genbank ChIL-2 The gene sequence (GI: 49169784) was used as a template. According to the degeneracy of codons and the preference of codons for proteins expressed by lactic acid bacteria, the codons were optimized and sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis, and the upstream and downstream were added Kpn I and HindⅢ Restriction site. artificially synthesized ChIL-2 The gene is connected to the pUC19 vector, that is, pUC19- ChIL-2 , using conventional enzyme digestion techniques, the application of restriction enzymes Kpn I and Hind Ⅲ Digestion of pUC19- ChIL-2 , obtained the target gene fragment with a size of 471bp ChIL-2 ,Such as figure 2 shown. Its base sequence is shown in the sequence table SEQ ID NO.2.

Embodiment 3

[0040] Example 3 SO7 Gene Linkage to pW425et

[0041] restriction endonuclease Nco I. Kpn Ⅰ Digest pMD18T- SO7 and pW425et, DNA Gel Extraction Kit Recovery SO7 After the gene fragment and the pW425et vector fragment, T4DNA ligase was used for ligation. pW425et, also known as pMG425et, is a vector that inserts the thyA gene (see Chinese patent CN1482247) into the vector pMG36e, uses thyA gene and erythromycin gene as selection pressure, and can shuttle and express between lactic acid bacteria and Escherichia coli.

[0042] Connection system:

[0043]

[0044] Reaction conditions: overnight connection at 16°C; routine CaCl the next day 2 law conversion E. coli DH5α competent, and screen for recombinant positive bacteria.

[0045] Extract recombinant positive bacteria plasmid, using Nco I. Kpn Ⅰ Carry out double enzyme digestion identification, such as image 3 As shown, the target bands with a size of about 4800bp and 651bp were cut out respectively, indicatin...

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Abstract

The invention discloses an immunological regulation type chicken E.tenella resistant recombinant lactobacillus and a preparation method thereof. A recombinant expression vector pW425et-SO7-ChIL-2 with a ChIL-2 gene and a coccidia SO7 gene is constructed by using a DNA (Deoxyribonucleic Acid) recombination technology according to the antigenicity of the SO7, the immunological regulation function of the IL-2 and the security of lactobacillus as an antigen presentation vector, and a corresponding lactic acid bacillus is prepared by using an electric conversion method. The recombinant lactobacillus can be used for remarkably increasing the daily gain of chicken, reducing the cecum lesion after worm attack and reducing the disordered bladder number and the relative disordered bladder yield of the cecum. Flow cytometry detection results prove that the recombinant lactobacillus can be used for regulating the CD4+ / CD8+T cell ratio and has a certain immunological regulation function as comparison with a control group. The preparation method and the production process are relatively simple; and the immunological regulation type chicken E.tenella resistant recombinant lactobacillus is orally taken by drinking water and is convenient for large-scale production and application and wide in application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an immunoregulatory anti-coccidiosis recombinant lactic acid bacterium and a preparation method thereof. Background technique [0002] Chicken coccidiosis is a common parasitic disease in the poultry industry, and it is very harmful to chicken production. It is common in the world, and the incidence rate of 15-50-day-old chicks is high, and the incidence rate of intensive chicken farms can reach 50%-70%, and the mortality rate can reach as high as 80% in severe cases. Can not recover for a long time. After the chicks were infected with Eimeria tenella, a large number of intestinal epithelial cells were damaged and hemorrhage, and severe cases caused death. At present, people have developed live vaccines and attenuated vaccines for chicken coccidia, such as the highly toxic Coccivac in the United States and the attenuated Eimerivax Plus vaccine in China. However, these ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/74C12N1/21A61K39/012A61P33/02C12R1/25C12R1/245C12R1/23
Inventor 杨桂连王春凤李博叶丽萍杨文涛郝凤奇
Owner JILIN AGRICULTURAL UNIV
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