Recombinant baculovirus for expressing heat-resistant HN protein of Newcastle disease virus as well as preparation method and application of recombinant baculovirus

A technology of recombinant baculovirus and Newcastle disease virus, which is applied in the fields of molecular biology and microbiology, can solve the problems of short immune protection period, incomplete inactivation, strong virulence, etc., and achieve improved thermal stability and thermal stability Good performance and cost reduction effect

Active Publication Date: 2021-01-19
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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Problems solved by technology

[0004] Subunit vaccines use molecular manipulation technology to clone pathogenic protective antigen genes into expression vectors to obtain antigenic proteins, and then make subunit vaccines, which are safe and can overcome the defects of traditional vaccines, such as the immune protection period of inactivated vaccines. Short, inactivation is not complete, resulting in the spread of the disease, and the phenomenon of virulence returning to strength caused by the mutation and recombination of the attenuated vaccine

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  • Recombinant baculovirus for expressing heat-resistant HN protein of Newcastle disease virus as well as preparation method and application of recombinant baculovirus
  • Recombinant baculovirus for expressing heat-resistant HN protein of Newcastle disease virus as well as preparation method and application of recombinant baculovirus
  • Recombinant baculovirus for expressing heat-resistant HN protein of Newcastle disease virus as well as preparation method and application of recombinant baculovirus

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preparation example Construction

[0026] The present invention also provides the preparation method of the recombinant baculovirus rAC-HN-N5 strain, which is characterized in that it comprises the following steps: a. the 49th, 269th, 293rd, 353rd and 353rd of the HN gene of the Newcastle disease virus LaSota strain 494 positions were mutated into E, S, E, Q and D respectively to obtain the mutated HN gene; b. insert the mutated HN gene into the middle of the LacZ gene of the baculovirus AcBac genome to obtain the whole genome DNA of the recombinant baculovirus; c . The recombinant baculovirus genome DNA and the AcBac baculovirus genome DNA were transfected into Sf9 insect cells to obtain the recombinant baculovirus rAC-HN-N5 strain.

[0027] The present invention also provides that the recombinant baculovirus rAC-HN-N5 strain can be used for highly expressing and purifying mutant HN protein. The protein has good thermal stability and can be used to prepare Newcastle disease heat-resistant subunit vaccines.

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Embodiment 1

[0029] Example 1 Construction and Rescue of Recombinant Baculovirus Expressing Newcastle Disease Virus HN Protein

[0030] According to the mutation protocol, four mutated HN gene sequences (HN-N1, HN-N3, HN-N5 and HN-N7) were obtained by gene synthesis. The mutation position of each gene is as figure 1 shown. The mutation scheme of HN-N1 is that the G at position 49 is mutated to E. The mutation scheme of HN-N3 is G at position 49 to E, R at position 269 to S and G at position 293 to E. The mutation scheme of HN-N5 is G at position 49 to E, R at position 269 to S, G at position 293 to E, R at position 353 to Q, and G at position 494 to D. The mutation scheme of HN-N7 is G mutation at position 49 to E, F mutation at position 151 to Y, R mutation at position 269 to S, G mutation at position 293 to E, R mutation at position 353 to Q, and mutation at position 494. G was mutated to D and V at position 495 was mutated to K. In the construction and rescue scheme of the recombin...

Embodiment 2

[0039] Example 2 Utilize recombinant baculovirus to express and purify mutant HN protein

[0040] The recombinant baculovirus was used to express and purify the mutant HN protein. The specific operation steps are as follows:

[0041] 2.1 Indirect immunofluorescence detection of expression of HN and its mutants

[0042] Sf9 cells were infected with baculoviruses rAc-HN, rAc-HN-N1, rAc-HN-N3, rAc-HN-N5, rAc-HN-N7 and rAcBac, respectively. 72h after infection, the expression of HN protein and its mutants in the cells was detected by indirect immunofluorescence. The result is as Image 6 As shown, specific green fluorescence appeared in rAc-HN, rAc-HN-N1, rAc-HN-N3, rAc-HN-N5 and rAc-HN-N7 infected cells, while rAcBac infected control cells No green fluorescence appeared, indicating that HN protein and its mutant proteins were expressed in Sf9 cells.

[0043] 2.2 SDS-PAGE electrophoresis and WB to detect the expression of HN and its mutants

[0044] sf9 cells were infected w...

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Abstract

The invention discloses a recombinant baculovirus for expressing heat-resistant HN protein of the Newcastle disease virus as well as a preparation method and application of the recombinant baculovirus. The virus strain is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: V202044. The preparation method of the ecombinant baculovirus comprises the following steps of carrying out point mutation on an HN gene of a Newcastle disease virus non-heat-resistant LaSota strain, including G49E, R269S,G293E, R353Q and G494D, inserting the mutated HN gene into a baculovirus genome, and carrying out virus rescue to obtain the recombinant baculovirus rAC-HN-N5 strain. The virus strain can efficientlyexpress mutant HN protein. Compared with wild-type HN protein of the LaSota strain, the mutant HN protein has the advantage that the thermal stability is obviously enhanced. The mutant HN protein expressed and purified by utilizing the recombinant baculovirus rAC-HN-N5 strain can be used for preparing a heat-resistant subunit vaccine for the Newcastle disease, so that the dependence of the vaccine on a cold chain is reduced, and a new thought is provided for the development of the subunit vaccine with high heat stability.

Description

technical field [0001] The invention relates to the fields of molecular biology and microbiology, in particular to a recombinant baculovirus expressing heat-resistant HN protein of Newcastle disease virus. More specifically, the present invention relates to the use of homologous recombination technology to carry out heat-resistant transformation on the Newcastle disease HN gene by way of point mutation, to obtain a recombinant baculovirus expressing the heat-resistant HN protein of Newcastle disease virus, and the virus strain in Application in the preparation of Newcastle disease heat-resistant subunit vaccine. Background technique [0002] Newcastle disease (Newcastle disease) is an acute, highly contagious infectious disease. Its pathogen is Newcastle disease virus. . NDV (Newcastle disease virus) belongs to the Paramyxoviridae family and a member of the genus Avian Mumps virus. It is a single-stranded, negative-strand, non-segmented RNA virus containing an envelope. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/45C12N15/866A61K39/17A61P31/14C12R1/92
CPCA61K39/12A61K2039/5256A61P31/14C07K14/005C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2760/18122C12N2760/18134
Inventor 商雨温国元李丽邵华斌罗青平王红琳罗玲张蓉蓉汪宏才张腾飞张文婷卢琴
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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