Method for preparing bivalent vaccine of newcastle disease virus La Sota strain and infectious bronchitis virus N-S multi-epitope protein

A technology for chicken Newcastle disease virus and dual vaccine, which is applied in the field of vaccine preparation, can solve the problems of low performance of the virus species, the need for concentration of infectious bronchitis virus inactivation, etc., and achieves the effects of good effect, prevention of chicken Newcastle disease virus, and reduction of immune stress.

Inactive Publication Date: 2015-04-29
TIANJIN RINGPU BIO TECH
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Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a method for preparing a double vaccine of chicken Newcastle disease virus La Sota strain and chicken infectious bronchitis virus N-S multi-epitope protein, so as to realize simultaneous immunity to chicken Newcastle disease virus infection and chicken kidney type infection bronchitis infection, and at the same time solve the technical problems of low performance of the virus seed used in the preparation of Newcastle disease vaccine and concentration of infectious bronchitis virus inactivation in the prior art

Method used

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  • Method for preparing bivalent vaccine of newcastle disease virus La Sota strain and infectious bronchitis virus N-S multi-epitope protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and protein expression of chicken kidney type infectious bronchitis N-S multi-epitope protein expression strain

[0026] The pGXα plasmid was extracted from the frozen pGXα-DH5α strain in our laboratory. The pGXα expression vector was constructed by the inventor. The vector was obtained by transforming the pPic9k expression vector of Pichia pastoris. The GAP gene fragment of the glyceraldehyde phosphate dehydrogenase promoter of Pichia pastoris was obtained, cloned into the vector pPic9k, and replaced the original AOX1 promoter, and the new expression vector constructed was named pGXα. A shuttle plasmid. And transformed into Escherichia coli DH5α, constructed the recombinant pGXα-DH5α strain, and stored in -80°C refrigerator.

[0027] Extract the pGXα plasmid and pUC57-N-S plasmid and digest them with NotI and XhoI to recover the pGXα expression vector and N-S gene. The linking system is as follows: T4DNA Ligase ligase 0.5 μL, 10×Buffer 1 μL, N-S...

Embodiment 2 2

[0033] The preparation of embodiment 2 dual vaccine

[0034] 1. Preparation of venom for vaccine production of Newcastle disease virus La Sota strain

[0035] The chicken embryos are selected from the eggs produced by healthy chicken flocks with good feeding and management, and each batch of chicken embryos must be strictly screened. A total of 4 items of external inspection: white shell, size, damage and dirty embryos, a total of 9 items of internal inspection: removal of sand shells, partial air chambers, free air chambers, inverted embryos, sterile eggs, terminated embryos, weak embryos, polluted embryos, Crack embryo. Qualified chicken embryos can be used as seedling making materials. Dilute Newcastle disease virus La Sota strain 1:10,000 times, inoculate 10-day-old SPF chicken embryos in the allantoic cavity, 0.1 mL per embryo, and incubate at a relative humidity of 60% to 65% and a temperature of 33 to 35°C. Turn the eggs, collect the allantoic fluid of the dead embry...

Embodiment 3 Embodiment 2

[0054] Example 3 evaluates the use effect of the dual vaccine prepared in Example 2

[0055] 80 21-day-old SPF chickens were randomly divided into 4 groups, of which 20 in group A were inoculated with a dose of 0.3ml, and group B was immunized with chicken Newcastle disease (La Sota) and infectious bronchitis (LDT3 kidney type) dual inactivated vaccine, Group C was immunized with chicken Newcastle disease (La Sota) and infectious bronchitis (H120 respiratory type) dual inactivated vaccine, and group D was the control group. 14 days after immunization, Beijing strain 1ml (10 4 ELD 50 ). The challenge dose of infectious bronchitis SD09 strain was 10 3.5 EID 50 . Observe 10 days, observe its morbidity and death situation, and all chickens are carried out necropsy on the 10th day, determine whether it is morbid. The specific results are shown in Table 1:

[0056] Table 1 Results of the immune effectiveness test of the dual vaccine

[0057]

[0058] The results show that...

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Abstract

The invention provides a preparation method of a bivalent vaccine by taking newcastle disease virus La Sota strain and infectious bronchitis virus N-S multi-epitope protein as antigen. To immunize 21-day-old chickens, the bivalent vaccine prepared by the method disclosed by the invention can be used for simultaneously preventing newcastle disease and nephrotropic infectious bronchitis virus infection, and the antibody level of the bivalent vaccine is higher than that of respectively immunizing by single vaccine, so that immunological stress is effectively relieved and immunity time is reduced.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to a method for preparing a chicken Newcastle disease virus La Sota strain and chicken infectious bronchitis virus N-S multi-epitope protein dual vaccine Background technique [0002] Newcastle disease, also known as Asian chicken plague, is a highly contagious, acute and severe infectious disease of chickens caused by Newcastle disease virus. Septicemia often presents, the main features are dyspnea, diarrhea, neurological disorders, mucosal and serosal hemorrhage. Chickens of all breeds and ages can be infected. At present, when chickens have certain immunity, Newcastle disease mainly appears in an atypical form, which needs to be paid attention to. [0003] Chicken infectious bronchitis virus nucleoprotein N can induce the body to produce high levels of antibodies, and the earliest time for antibody production is that the ELISA titer is higher than that of antibodies...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61P31/14A61K39/17
Inventor 陈冰李亚杰郁宏伟杨保收梁武
Owner TIANJIN RINGPU BIO TECH
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