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Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof

A technology of Newcastle disease virus and recombinant plasmid, which is applied in the field of recombinant plasmid, recombinant gene VII Newcastle disease virus and its cultivation, and can solve the problems of high content of miscellaneous proteins, safety and stability to be improved, production waste pollution, etc.

Pending Publication Date: 2021-04-09
ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the course of long-term practice, people have found that there are risks of exogenous virus contamination in the production and application of chicken embryo and spinner vaccines, the risk of contamination from production waste, the risk of batch-to-batch variability, and the risk of immune side effects due to high content of miscellaneous proteins. Both stability and stability need to be improved

Method used

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  • Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof
  • Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof
  • Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Materials used in the experiment:

[0048] Materials: Nucleic Acid Extraction Kit (Taktara 9767); Reverse Transcription Kit (Takara 023A); Gel Recovery Kit (OMEGA D2500-02); High Fidelity Enzyme Prime STAR MAX (Takara R045A); Gene Recombination Kit (Vazyme C113 ); DH5α competent cells (Takara 9057); common [CR enzyme Premix-Taq (Takara RR902A); SmaI, PstI endonuclease (NEB); transfection kit (Invitrogen 15338500); BHK-21 cells, pBR322-FDHN3 Plasmids are preserved by Huanong (Zhaoqing) Bioindustry Technology Research Institute Co., Ltd.

[0049] 1. Construction of recombinant plasmid pBR322-FDHN3-S1 of gene VII Newcastle disease virus expressing infectious bronchitis virus (IBV) S1 protein gene.

[0050] (1) Preparation of S1 gene fragment of IBV

[0051] ① Take chicken embryo allantoic fluid containing IBV virus, and extract total RNA according to the steps of the nucleic acid extraction kit;

[0052] ② Use a reverse transcription kit to obtain viral cDNA;

[0053] ...

Embodiment 2

[0104] Establishment of rFDHN3-S1 recombinant Newcastle disease virus culture method in full suspension MCDK cell line

[0105]Materials: MDCK cells, rFDHN3-S1 recombinant Newcastle disease attenuated vaccine candidate strain, all preserved by Huanong (Zhaoqing) Institute of Bioindustry Technology; CD MDCK 244 serum-free medium, purchased from Gansu Jianshun Biotechnology Co., Ltd.; TPCK trypsin , purchased from sigma

[0106] (1) Subculture of MDCK cells: Add the subcultured MDCK cells into a Erlenmeyer shaker flask containing 100 mL of medium, and place in CO 2 Shaking incubator, set the temperature at 37°C and rotate at 120rpm / min, when the cell density reaches 8.0~9.0×10 6 cells / mL, take an appropriate cell suspension and add it to fresh medium, so that the total volume of the culture system is 100ml, and the cell density is about 1.5×10 6 cells / mL, cultured to a cell density of 8.0-9.0×10 6 cells / mL, repeat the above process.

[0107] (2) Establishment of the culture ...

Embodiment 1

[0116] Experimental procedure

[0117] (1) Take the cell density to reach 8.0~9.0×10 6 cells / mL MDCK cell suspension, diluted in proportion to a cell density of 3.0×10 6 cells / mL, culture system 50ml;

[0118] (2) Take the virus titer as 10 8.0 EID50 / 0.1ml rFDHN3-S1 recombinant Newcastle disease virus solution was added to the culture system in proportion until the virus content was 0.0001MOI, and TPCK trypsin was added at 4mg / L at the same time, and placed in CO 2 Shake the incubator, set the temperature at 37°C, and cultivate at a speed of 120rpm / min.

[0119] (3) Samples were taken at 24h, 48h, 72h, and 96h of culture, and the HA titer was detected by the micromethod. When HA≥1:128, the virus titer (EID50) was calculated using the Reed-Muench method.

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Abstract

The invention belongs to the field of biology, and discloses a recombinant plasmid pBR322-FDHN3-S1. The sequence of the recombinant plasmid pBR322-FDHN3-S1 is shown as a sequence table SEQ ID NO: 1. The recombinant plasmid is based on a VII subtype NDV strain DHN3 strain with strong toxicity, an F gene of a Lasota attenuated strain is used for replacing an F gene of the DHN3 strain, an S1 gene is inserted, and the recombinant plasmid has immunogenicity for II-type and VII-type Newcastle diseases and IBV. Meanwhile, the invention also discloses a recombinant virus and a high-titer culture method for the virus.

Description

technical field [0001] The invention relates to the biological field, in particular to a recombinant plasmid, a recombinant gene VII type Newcastle disease virus and a culture method thereof. Background technique [0002] Chicken Newcastle disease (ND) and chicken infectious bronchitis (IB) are acute and highly contagious viral diseases of chickens caused by Newcastle disease virus (NDV) and chicken infectious bronchitis virus (IBV) respectively. Chickens of different ages, sexes and breeds are susceptible, and can cause diseases such as the respiratory system, digestive system, and reproductive system in the flock, and even death. In poultry production, the mixed infection of the two viruses often occurs, which can further aggravate the disease situation and seriously affect the health and production efficiency of chicken flocks. The above-mentioned diseases must be strictly guarded against, and vaccine prevention and control is currently the most important means. With th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/50C12N15/45C12N7/01C12N5/071C12R1/93
CPCC12N15/85C07K14/005C12N7/00C12N5/0686C12N2800/107C12N2760/18121C12N2760/18122C12N2760/18152C12N2770/20022C12N2770/20051C12N2760/18151C12N2509/00C12N2500/90
Inventor 叶俊贤方倪冉郑航辉陈杏桃杨小云董楠
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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