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pMG36e pgsA gp85 recombinant plasmid-containing genetically engineered bacterium

A pmg36e-pgsa-gp85, genetically engineered bacteria technology, applied in the field of biology, can solve the problems of complex antigen structure, difficult attenuated vaccines, destroying antibodies, etc., and achieve the effect of increasing average daily weight gain, reducing risks, and improving ability

Inactive Publication Date: 2017-12-29
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the pollution of the breeding environment, it is impossible to completely control ALV-J infection only by purification.
In recent years, many experts and scholars have also tried to develop ALV-J inactivated vaccines and attenuated vaccines, but because the inactivated virus has also destroyed its ability to induce antibodies, and the antigenic structure of the virus is complex, it is difficult to obtain an ideal vaccine. Attenuated vaccines are very difficult

Method used

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  • pMG36e pgsA gp85 recombinant plasmid-containing genetically engineered bacterium
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  • pMG36e pgsA gp85 recombinant plasmid-containing genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 Amplification of gp85 gene and pgsA gene

[0064] (1) Design specific primers:

[0065] The PCR primer 5'-C was used in the PCR amplification of the target fragment pgsA GAGCTC GCGAACTGAGCTTTCATGAAAAG-3', its nucleotide sequence is shown in SEQ ID NO.1; 5'-CTAG TCTAGA CTATGATCAATATCAAACGTCA-3', the nucleotide sequence of which is shown in SEQ ID NO.2;

[0066] The PCR primer 5'-TCA was used in the PCR amplification of the target fragment gp85 TCTAGA GGGAGTTCATCTGTTG-3', its nucleotide sequence is shown in SEQ ID NO.3 and 5'-TCC AAGCTT ATTAGCGCCTGCTAC-3', the nucleotide sequence of which is shown in SEQ ID NO.4;

[0067] Utilize the above primers to amplify the gp85 envelope glycoprotein gene from the J subgroup avian leukosis virus env gene; amplify the pgsA anchor protein gene from the pgsA, pgsB and pgsC genes in the whole gene of Bacillus subtilis; the results are as follows figure 2 shown;

[0068]PCR amplification system and conditions of gp8...

Embodiment 2

[0075] The PCR products of gp85 and pgsA gene obtained in step (1) are respectively connected to two separate pMD18-T vectors;

[0076] The pMD18-T vector was ligated with the purified and recovered two target gene fragments respectively. The ligation system was 0.5 μl of pMD18-T vector, 5.0 μl of Solution Ⅰ, and 4.5 μl of purified PCR product. Mix the ligation product evenly, centrifuge slightly to remove air bubbles, place it at 16°C for 8-10 hours, and then store it at 4°C for future use.

Embodiment 3

[0077] Example 3 Obtaining of recombinant plasmid pMG36e-pgsA-gp85

[0078] Recycling of expression vector pMG36e and gp85 target gene fragment by double enzyme digestion

[0079] The expression vector pMG36e and the target gene gp85 fragment were digested with Hind Ⅲ and Xba Ⅰ respectively. Use 50 μl enzyme digestion system: 10×M Buffer, 6 μl; Xba Ⅰ and Hind Ⅲ 3 μl each; gp85 / pMG36e 15 μl; add ddH 2 O 23 μl. Mix the reactants according to the above system, and put them in a water bath at 37°C for 3h. The pMG36e vector fragment and the target fragment gp85 were purified and recovered with a gel recovery kit. After product recovery and purification, 1% agarose gel electrophoresis was used to observe the recovery situation.

[0080] Ligation transformation of target gene gp85 and expression vector pMG36e

[0081] a. Ligation: Ligate the enzyme-digested vector fragment recovered from the gel with the enzyme-digested target fragment. Use 20 μl ligation system: pMG36e 8 μl; g...

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Abstract

The invention provides a pMG36e pgsA gp85 recombinant plasmid-containing genetically engineered bacterium. A lactobacillus plantarum engineering bacterial strain which can express fusion protein pgsA gp85 is constructed by inserting a gene gp85 and a gene pgsa into Escherichia coli-lactobacillus shuttle type expression vector pMG36e to obtain a recombinant vector pMG36e-pgsA gp85, and then electrically converting the recombinant vector pMG36e-pgsA gp85 into the lactobacillus plantarum engineering bacterial strain, wherein the bacterial strain can express to obtain corresponding pgsA gp85 fusion protein; the fusion protein can be widely applied to oral immunization, the average daily weight gain of chickens in each period can be greatly improved, and moreover, the resistance of the chickens to ALV-J (Avian Leukosis Virus subgroup) infection can be improved, so that the culture benefit is beneficially increased, and the ALV-J infecting risk of chicken bodies is beneficially reduced.

Description

technical field [0001] The invention relates to the field of biology, and specifically provides a genetic engineering bacterium containing pMG36e-pgsA-gp85 recombinant plasmid. Background technique [0002] J subgroup avian leukemia virus (Avian Leukosis Virus subgroup J, ALV-J) was first isolated from broiler chickens in 1988, mainly causing myeloid cell hyperplasia and nephroma in chickens. The disease has spread rapidly to many countries, causing huge losses to the poultry industry in the world. [0003] At present, there is no commercialized vaccine for the prevention and treatment of ALV-J infection. Foreign countries mainly control ALV-J by purifying breeder flocks. However, since the breeding environment has been polluted, it is impossible to completely control ALV-J infection only by purification. In recent years, many experts and scholars have also tried to develop ALV-J inactivated vaccines and attenuated vaccines, but because the inactivated virus has also destr...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K39/21A61P31/14C12R1/25
CPCA61K39/12A61K2039/523A61K2039/542A61K2039/552C07K14/005C12N2740/11022C12N2740/11034
Inventor 刘建柱王胜华刘永夏成子强赵鹏张普郭慧君
Owner SHANDONG AGRICULTURAL UNIVERSITY
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