Recombinant vector, recombinant baculovirus and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a recombinant vector, a recombinant baculovirus and a preparation method and application of the recombinant vector and the recombinant baculovirus. According to the method for preparing the recombinant vector, pFastBac Dual is adopted as a vector to prepare a recombinant plasmid vector inserted into a VP2 gene of an avian bursal disease virus classic strain, and the recombinant plasmid vector is named as pDual-V1; the method comprises the following steps: preparing a recombinant plasmid vector into which VP2 genes of a classical strain and a variant strain of the avian bursal disease virus are inserted at the same time, and naming the recombinant plasmid vector as pDual-V1-V2; the method comprises the following steps: preparing a recombinant plasmid vector inserted into a group I type 4 fowl adenovirus Fiber-2 gene, and naming the recombinant plasmid vector as pDual-F2; the method comprises the following steps: preparing a recombinant plasmid vector into which a Fiber-2 gene and an avian reovirus sigma C gene sequence are inserted at the same time, wherein the recombinant plasmid vector is named as pDual-F2-sigma C; carrying out double enzyme digestion on the pDual-V1-V2, recovering small fragments, carrying out double enzyme digestion on the pDual-F2-sigma C, recovering large fragments, and then carrying out mixed enzyme ligation. The recombinant baculovirus can express four antigens at the same time, and effective preparation of a multi-combined vaccine is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant vector, a recombinant baculovirus and a preparation method and application thereof. Background Art

[0002] Avian reoviruses (ARVs) belong to the genus Orthoreovirus of the family Reoviridae, and their genome is double-stranded segmented RNA. ARVs can affect a variety of poultry, including chickens, ducks, and geese. Broilers infected with ARVs show clinical symptoms such as tenosynovitis, myocarditis, immunosuppression, and developmental retardation syndrome. In its genome, σC encoded by the S1 gene is a capsid protein. The protein participates in cell adsorption and can induce the body to produce specific neutralizing antibodies. It is the main antigenic determinant of ARVs. Its mutation can lead to changes in viral pathogenicity and plays an important role in ARV infection and pathogenesis.

[0003] Fowl adenovirus (FAdV) belongs to the genus Avian adenovirus of the family Avian Adenoviridae. According to antigenic characteristics, fowl adenovirus can be divided into three groups: I, II, and III. Group I can be divided into five species and twelve serotypes, which can cause clinical and pathological symptoms in a variety of poultry, including pericardial effusion and inclusion body hepatitis. Group I adenovirus has a more complex transmission mode, spreads and spreads rapidly, is more common in summer, and has a high mortality rate. The research and development of adenovirus vaccines is imperative.

[0004] Infectious bursal disease (IBD) is an acute, highly contagious disease caused by infectious bursal disease virus (IBDV). The virus mainly attacks the lymphoid tissues of chicks and young chickens, especially central immune organs such as the bursa of Fabricius, causing severe pathological damage to the bursal tissues, leading to severe immunosuppression in the body, which can easily lead to secondary infections or failure of multiple vaccine immunizations.

[0005] VP2, the main structural protein of infectious bursal disease virus of chickens, is also the main component of the viral nucleocapsid. It has a conformation-dependent (discontinuous) neutralizing antigenic determinant cluster. The induced neutralizing antibodies can passively protect the host from IBDV infection. It is a host protective antigen of IBDV. Its research plays an important role in the genetic variation and immune prevention and control of IBDV.

[0006] At present, the prevention of avian reovirus, avian adenovirus, and avian bursal disease virus infection is still mainly based on traditional attenuated vaccines and inactivated vaccines. Due to differences in preparation systems, the immune efficacy and biosafety of the vaccines are also different. How to provide protection against these three viruses at the same time still needs further research. Summary of the invention

[0007] One of the purposes of the present invention is to provide a method for simultaneously and efficiently expressing the VP2 antigen protein of the classical strain of avian bursal disease virus, the VP2 antigen protein of the variant strain, the Fiber-2 antigen protein of the group I type 4 avian adenovirus and the ARV-σC antigen protein of the avian reovirus.

[0008] The present invention provides a method for preparing a recombinant vector, which uses pFastBac Dual as a vector, inserts the VP2 gene of the classical strain of avian bursal disease virus into the restriction sites of EcoRI and XbaI restriction endonucleases of the vector, and obtains a recombinant plasmid vector with the VP2 gene of the classical strain of avian bursal disease virus inserted therein, which is named pDual-V1; The VP2 gene of the variant strain of avian bursal disease virus and the pDual-V1 were double-digested with XhoI and KpnI, respectively, and then ligated by enzymes to obtain a recombinant plasmid vector into which the VP2 gene of the classical strain of avian bursal disease virus and the VP2 gene of the variant strain of avian bursal disease virus were simultaneously inserted, and was named pDual-V1-V2; Inserting the Fiber-2 gene of group I type 4 fowl adenovirus into the EcoRI and XbaI restriction sites of the vector to obtain a recombinant plasmid vector with the Fiber-2 gene of group I type 4 fowl adenovirus inserted therein, named pDual-F2; The avian reovirus σC gene sequence and the pDual-F2 were double-digested with XhoI and KpnI, respectively, and then ligated to obtain a recombinant plasmid vector into which both the group I type 4 avian adenovirus fiber-2 gene and the avian reovirus σC gene sequence were inserted, and the vector was named pDual-F2-σC. The pDual-V1-V2 is subjected to double digestion with PmeI / AvrII to recover a small fragment (a relatively shorter fragment of the two fragments obtained after double digestion); the pDual-F2-σC is subjected to double digestion with SpeI / NruI to recover a large fragment (a relatively longer fragment of the two fragments obtained after double digestion), and then mixed enzyme ligation is performed to obtain a recombinant plasmid vector containing the VP2 gene of the classical strain of avian bursal disease virus, the VP2 gene of the variant strain of avian bursal disease virus, the Fiber-2 gene of the group I type 4 avian adenovirus, and the σC gene of the avian reovirus, which is named pDual-V1-V2-F2-σC; The sequence of the VP2 gene of the classical strain of avian bursal disease virus is shown in SEQ ID No. 1; The sequence of the VP2 gene of the avian bursal disease virus variant is shown in SEQ ID No. 2; The sequence of the Fiber-2 gene of the group I type 4 avian adenovirus is shown in SEQ ID No. 3; The sequence of the avian reovirus σC gene is shown in SEQ ID No.4; Preferably, the primer sequences used to amplify the VP2 gene of the classical strain of avian bursal virus, the VP2 gene of the variant strain of avian bursal virus, the Fiber-2 gene of the group I type 4 avian adenovirus and the σC gene of the avian reovirus are as shown in SEQ ID No. 5-12.

[0009] The recombinant baculovirus prepared by the recombinant vector of the present invention can simultaneously and efficiently express the VP2 antigen protein of the classical strain and variant strain of avian bursal disease virus, the Fiber-2 antigen protein of group I type 4 avian adenovirus, and the ARV-σC antigen protein of avian reovirus. This is due to the specific optimization and combination order of each gene sequence in the recombinant vector of the present invention. If other gene sequences or optimization methods are used, or the connection order of each gene is adjusted, the expression of each protein will be affected, and the effect of simultaneously and efficiently expressing all antigen proteins cannot be obtained.

[0010] The present invention also provides a method for preparing a recombinant baculovirus, comprising: (1) Clone the DNA molecule into Escherichia coli DH10 Bac competent cells and screen and identify the recombinant baculovirus Bacmid; The DNA molecule comprises a sequentially connected Fiber-2 gene of group I type 4 avian adenovirus, a VP2 gene of a variant strain of avian bursal virus, a VP2 gene of a classical strain of avian bursal virus, and a σC gene of an avian reovirus; the sequences of the Fiber-2 gene of group I type 4 avian adenovirus, the VP2 gene of a variant strain of avian bursal virus, the VP2 gene of a classical strain of avian bursal virus, and the σC gene of an avian reovirus are as described above; (2) transfecting SF9 cells with the recombinant baculovirus Bacmid obtained in step (1), and harvesting the recombinant baculovirus after culture; Preferably, in step (1), the recombinant vector prepared by the above method is used to transform Escherichia coli DH10 Bac competent cells, so as to clone the DNA molecule into the Escherichia coli DH10 Bac competent cells.

[0011] The present invention preferably uses pFastBac Dual as the vector of the above-mentioned DNA molecule, which can be used to prepare recombinant baculovirus Bacmid after forming a recombinant vector.

[0012] The present invention also provides a recombinant baculovirus, which is prepared by the above method; or, the deposit number of the recombinant baculovirus is CCTCC NO: V202448.

[0013] The present invention provides an rBac-V1-V2-F2-σC genetically engineered strain (Autographa californica multiplenuclecopolyhedrovirus) constructed according to the above method. The strain has been deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC NO: V202448, and deposit date April 23, 2024.

[0014] The baculovirus strain rBac-V1-V2-F2-σC prepared by the method provided by the present invention can be rapidly propagated on SF9 / High5 cells, and a higher protein titer can be obtained in a shorter time, and can be used to prepare a triple or multi-unit subunit vaccine of avian bursal classic strain / variant strain-group I type 4 avian adenovirus-avian reovirus.

[0015] The present invention also provides a DNA molecule as described above.

[0016] The present invention also provides a DNA molecule, whose nucleotide sequence is shown as SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4.

[0017] The present invention also provides a biological material containing the above DNA molecule, wherein the biological material is an expression cassette, a recombinant vector, a transgenic cell line, a recombinant bacterium or a recombinant virus.

[0018] The present invention also provides a method for preparing the virus culture fluid of the recombinant baculovirus, comprising: (1) Cultivating whole suspension cells; preferably, the whole suspension cells are insect cells, cultured in a whole suspension serum-free medium, and more preferably, the insect cells are High5 cells; (2) inoculating the above-mentioned recombinant baculovirus and conducting suspension culture; (3) Harvesting virus culture fluid; Preferably, in step (2), when the recombinant baculovirus according to claim 3 is inoculated, the growth density of the whole suspension cells is 5.0×10 6 -7.0×10 6 cells / mL, add medium when inoculating the virus, and dilute the total suspended cell count to 2.8×10 6 -3.0×10 6 cells / mL; and / or, the virus inoculation amount is MOI=0.5-2, and the culture conditions are: temperature 27±1°C, dissolved oxygen 55-65% (preferably, 55-60%), stirring speed 80 rpm, pH 6.0-6.2; Preferably, in step (3), when the viability of the whole suspension cells drops below 70% and the density drops to 2×10 6 When the concentration was below 10 cells / mL, the culture was terminated, and the whole suspended cells were disrupted and the virus culture fluid was collected.

[0019] The present invention provides the use of full suspension cells (such as full suspension High5 cells) in full suspension culture of baculovirus. The baculovirus may be AcMNPV, preferably the above-mentioned recombinant baculovirus of the present invention.

[0020] By using the Bac-to-Bac baculovirus expression system, the method of the present invention can simultaneously and efficiently obtain the avian bursal virus classic strain VP2, variant strain VP2 antigen protein, group I type 4 avian adenovirus Fiber-2 antigen protein and avian reovirus ARV-σC antigen protein, thereby greatly improving the protein expression efficiency while ensuring the quality of the antigens. By emulsification immunization, the immunized chickens can simultaneously resist the attacks of the bursal virus classic strain, the bursal virus super strain, the bursal virus variant, group I type 4 avian adenovirus and avian reovirus.

[0021] In the present invention, it is preferred that cells are transfected with the recombinant baculovirus containing the above DNA molecule, and then cultured, passaged three times, and the cells are completely frozen and thawed twice, and the supernatant is collected by centrifugation to obtain a recombinant baculovirus-expressed protein mixture.

[0022] The method provided by the present invention is suitable for small-scale and 1000L large-scale production of the above-mentioned recombinant baculovirus strains, and provides a technical basis for further efficient production of avian bursal classic strain / variant strain-group I type 4 avian adenovirus-avian reovirus triple bivalent and multi-valent genetic engineering subunit vaccines.

[0023] In the method, the full suspension serum-free medium used can be any full suspension serum-free medium suitable for culturing full suspension cells (such as full suspension High5 cells). Before inoculation, the full suspension cells are preferably resuscitated and cultured in a step-by-step manner. The above culture can be carried out in a 1000L bioreactor.

[0024] The present invention also provides a virus culture solution, which is prepared by the above method.

[0025] The virus culture fluid of the present invention can be inactivated (for example, inactivated in a formaldehyde solution at 37°C for 24 hours) and then emulsified to prepare a bivalent multi-unit vaccine of a classical strain / super strain / variant strain of avian bursal disease virus, a group I type 4 avian adenovirus, and an avian reovirus.

[0026] The present invention also provides the method for preparing the recombinant vector or the method for preparing the recombinant baculovirus or the recombinant baculovirus or DNA molecule or biological material or the method for preparing the virus culture fluid of the recombinant baculovirus or the use of the virus culture fluid in preparing vaccines.

[0027] The subunit protein prepared by this method has a high titer and does not need to be concentrated when preparing the vaccine. Animal experiments have confirmed that it has high immunogenicity and safety, and can help immunize chickens against attacks of avian bursal classic strains, variant strains, super strains, group I type 4 avian adenovirus and avian reovirus.

[0028] The present invention also provides a vaccine, which comprises the above virus culture fluid.

[0029] The vaccine of the present invention can prevent and / or treat infection by classical strains and / or super strains and / or variant strains of avian bursal disease virus and / or infection by group I type 4 avian adenovirus and / or avian reovirus, and its active ingredient is the expression product of the recombinant baculovirus strain.

[0030] The beneficial effects of the present invention are at least: The recombinant baculovirus prepared by the recombinant vector provided by the present invention can simultaneously and efficiently express four effective antigen proteins in one preparation system, and obtain a high amount of avian bursal disease virus classical strain VP2, variant strain VP2 antigen protein, group I type 4 avian adenovirus Fiber-2 antigen protein and avian reovirus ARV-σC antigen protein in a relatively short time. The preparation process is simple, suitable for preparing combined vaccines, and has good immunogenicity and safety as proved by animal immunization and challenge tests.

[0031] In addition, in the process of testing the semi-finished products of the traditional group I type 4 avian adenovirus antigen, the TCID50 test method used is relatively cumbersome and cannot directly reflect the problem of effective antigen content. The group I type 4 avian adenovirus Fiber-2 antigen prepared by the present invention can be detected by the intuitive method of agar diffusion test, and a clear and observable precipitation line can be formed, which can be used as a reference for the amount of antigen protein used in vaccine preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The PCR amplification diagrams of FADV-Fiber2, ARV-σC gene, VP1 gene and VP2 gene in Example 1 of the present invention are shown in the figure below. Lane 1 on the left: Fiber2; Lane 2: σC; Lane 3: DL2000. Lane 1 on the right: DL2000; Lane 2: V1; Lane 3: V2.

[0033] Figure 2 This is a diagram of the pDual-V1-V2-F2-σC sequence construction in Example 1 of the present invention.

[0034] Figure 3 The morphological observation results of SF9 cells in Example 2 of the present invention are shown in the left figure: the microscopic morphology of healthy SF9 cells cultured in serum-free culture for 72 hours (100×); the microscopic morphology of SF9 cells cultured in serum-free culture for 72 hours after inoculation with recombinant baculovirus rBac-V1-V2-F2-σC (100×).

[0035] Figure 4 This is the SDS-PAGE identification diagram of the recombinant baculovirus strain rBac-V1-V2-F2-σC in Example 3 of the present invention. Lane 1 is the middle molecular weight protein marker; Lane 2 is the P3 generation pDual-V1-V2-F2-σC virus seed culture fluid, and Lane 3 is the baculovirus fluid negative control.

[0036] Figure 5 The Western-Blot results of the recombinant baculovirus strain rBac-V1-V2-F2-σC in Example 3 of the present invention are shown in Figure 1. Lane 1 is the protein sample prepared by the present invention, and lane 2 is the protein molecule marker.

[0037] Figure 6 This is the mass spectrometry analysis result of the recombinant baculovirus strain rBac-V1-V2-F2-σC in Example 3 of the present invention.

[0038] Figure 7 The results of observation of High5 cell morphology in Example 4 of the present invention are shown in the left figure. The left figure shows the microscopic morphology of fully suspended High5 healthy cells cultured for 48 hours. The right figure shows the microscopic morphology of fully suspended High5 healthy cells cultured for 48 hours after inoculation with the recombinant baculovirus rBac-V1-V2-F2-σC.

[0039] Figure 8 Schematic diagram of the production process of recombinant baculovirus rBac-V1-V2-F2-σC in High5 suspension cell culture in a 1000 L scale bioreactor in Example 4 of the present invention.

[0040] Fig. 9 This is a growth and proliferation trend curve of healthy cells in the 50L, 250L, and 1000L bioreactor High5 in Example 4 of the present invention.

[0041] Fig.10 These are the results of observation of the morphology of High5 cells fully suspended in a 1000 L bioreactor in Example 4 of the present invention. The left figure shows the microscopic morphology of healthy High5 cells fully suspended in a 1000 L bioreactor after culture for 72 hours; the right figure shows the microscopic morphology of healthy High5 cells fully suspended in a 1000 L bioreactor after inoculation with the recombinant baculovirus rBac-V1-V2-F2-σC and culture for 72 hours.

[0042] Fig.11 This is a graph showing the agar titer detection of FADV (left) and IBDV (right) antigens prepared in the 1000 L bioreactor in Example 4 of the present invention.

[0043] Fig.12 This is an anatomical diagram of the diseased and dead chickens in the control group of the IBDV challenge test in Example 8 of the present invention.

[0044] Fig.13 This is an anatomical diagram of chickens that became ill and died in the control group of the FADV challenge test in Example 8 of the present invention. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources or prepared according to conventional methods in the art. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions. The full suspension serum-free medium was purchased from Suzhou Womei Biological Co., Ltd.

[0047] Example 1 Construction of the recombinant vector pDual-V1-V2-F2-σC 1. In this example, the VP2 gene of the classical strain of avian bursal disease virus was optimized for codon adaptability and then added with a 6×His tag (SEQ ID No. 1, using the IBDV / GenBank: AAG23824.1 protein sequence as a template, after codon adaptability optimization and synthesis, and amplified with primers VP2-1-F and VP2-1-R), and cloned into the EcoRI and XbaI restriction endonuclease sites of the pFastBac Dual vector to obtain a recombinant plasmid vector inserted with the VP2 gene of avian bursal disease virus, named pDual-V1.

[0048] 2. The VP2 gene of the variant strain of avian bursal virus (SEQ ID No. 2, using the IBDV / GenBank: UCF77630.1 protein sequence as a template, after codon adaptability optimization and synthesis, and amplified with primers VP2-2-F and VP2-2-R) and the pDual-V1 prepared in step 1 were double-digested with XhoI and KpnI, respectively, and then enzyme-ligated to obtain a recombinant plasmid vector into which the VP2 gene of the classical strain of avian bursal virus and the VP2 gene of the variant strain of avian bursal virus were simultaneously inserted, and was named pDual-V1-V2.

[0049] 3. The group I type 4 fowl adenovirus Fiber-2 gene was codon-optimized (SEQ ID No. 3, using the FADV / GenBank: AYU58968.1 protein sequence as a template, codon-optimized and synthesized, and amplified with primers Fiber2-F and Fiber2-R) and cloned into the EcoRI and XbaI restriction sites of the pFastBac Dual plasmid to obtain a plasmid vector with the group I type 4 fowl adenovirus Fiber-2 gene inserted, named pDual-F2.

[0050] 4. The avian reovirus σC gene sequence, after codon adaptability optimization (SEQ ID No. 4, using the σC protein sequence in ARV / GenBank: AB914766.1 as a template, after codon adaptability optimization and synthesis, amplified with primers σC-F and σC-R), was double-digested with XhoI and KpnI with the pDual-F2 plasmid obtained in step 3, and then enzyme-ligated to obtain a plasmid vector simultaneously inserted with the group I type 4 avian adenovirus Fiber-2 gene and the avian reovirus σC gene sequence, named pDual-F2-σC.

[0051] 5. Take the pDual-V1-V2 plasmid obtained in step 2, perform double digestion with PmeI / AvrII on it, and recover the small fragment; take the pDual-F2-σC plasmid obtained in step 4, perform double digestion with SpeI / NruI, respectively, recover the large fragment, and then perform mixed enzyme ligation to obtain a plasmid vector containing the VP2 gene of the classical strain of avian bursal disease virus, the VP2 gene of the variant strain of avian bursal disease virus, the Fiber-2 gene of group I type 4 avian adenovirus, and the σC gene of avian reovirus, and name it pDual-V1-V2-F2-σC.

[0052] The pFastBac Dual vector (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) was preserved in this laboratory. The electrophoresis diagram of the above DNA fragments is shown in Figure 1 The sequence diagram of pDual-V1-V2-F2-σC is shown in Figure 2, where F represents Fiber-2 gene, V2 represents VP2 gene, V1 represents VP1 gene, and C represents σC gene. The primer information used to amplify the above genes is shown in Table 1.

[0053] Table 1 Amplification primers for each fragment (SEQ ID No. 5-12) Example 2 Preparation of recombinant baculovirus rBac-V1-V2-F2-σC In this example, the above recombinant plasmid pDual-V1-V2-F2-σC was transformed into DH10 Bac competent cells, and after the resistance was restored in LB liquid medium without resistance for 2 hours, it was coated on a LB solid plate containing tetracycline, gentamicin, kanamycin, IPTG and X-Gal, and inverted at 37°C for 24-48 hours for blue-white screening, and a single white colony was picked for PCR testing. The positive clone was picked and inoculated in LB liquid medium containing tetracycline, gentamicin and kanamycin for shaking and culturing overnight, and the recombinant baculovirus Bacmid was extracted and named rBac-V1-V2-F2-σC.

[0054] SF9 cells in the logarithmic growth phase were cultured at a rate of 5.0×10 5 -8.0×10 5 After dilution at 1.5447 W / mL, the cells were plated in a 6-well plate and transfected with Lipofectamine 3000 transfection reagent and recombinant baculovirus Bacmid rBac-V1-V2-F2-σC. The cells were cultured at 27°C for 5-7 days until the cell viability was lower than 40%. The cell supernatant was collected and centrifuged at 5000 rpm for 3 min. The supernatant was collected, which was the P0 strain of the recombinant baculovirus rBac-V1-V2-F2-σC (the deposit number is CCTCC NO: V202448).

[0055] The P0 strain of the recombinant baculovirus rBac-V1-V2-F2-σC was inoculated into SF9 cells in the logarithmic growth phase at a ratio of 1:50 and cultured in a 27°C incubator for 5-7 days until the cell viability was less than 40%. The cell supernatant was collected to obtain the P1 strain of the recombinant baculovirus rBac-V1-V2-F2-σC. The P3 strain was obtained by passage using the same method and TCID 50 Detection, rBac-V1-V2-F2-σCP3 generation virus titer is 10 -7.5 / 0.1mL.

[0056] The morphology of SF9 healthy cells cultured without serum for 72 hours under the microscope is shown in Figure 3Left: The microscopic morphology of serum-free adherent SF9 cells inoculated with recombinant baculovirus rBac-V1-V2-F2-σC after 72 hours of culture. Figure 3 Right picture.

[0057] Example 3 Expression and identification of recombinant proteins in insect cells High5 Will grow to 5.0×10 6 -6.0×10 6 cells / ml High5 cells were diluted to 2.8×10 cells / ml using full suspension serum-free medium. 6 -3.0×10 6 cells / ml, inoculated with the P3 generation rBac-V1-V2-F2-σC recombinant baculovirus obtained in Example 2, cultured for 5 days, until the cell viability decreased to less than 40%, the cells were broken by freeze-thaw method, and the IBDV-VP2 protein and FADV-Fiber2 protein therein were tested by agar amplification, and the agar amplification titers of VP2 protein and Fiber2 protein could reach ≥1:32 at the same time; the SDS-PAGE identification figure of the recombinant baculovirus strain rBac-V1-V2-F2-σC is shown in Figure 4 The ARV-σC protein was tested by serum ELISA after immunization, and the antibody positive rate could reach 100%.

[0058] To further identify the expression of the four proteins, the cell culture fluid obtained after 5 days of infection with the P3 virus was subjected to Western Blot analysis. Figure 5 As shown, a clear band can be seen at 50KD; the sample was subjected to mass spectrometry analysis, and the analysis and comparison results successfully matched the IBDV-VP2 proteins of different strains, indicating that rBac-V1-V2-F2-σC can successfully express the VP2 protein of the classical strain / variant strain of avian bursal disease. The mass spectrometry analysis results of the recombinant baculovirus strain rBac-V1-V2-F2-σC are shown in Figure 6 .

[0059] Example 4 Establishment of a 1000L bioreactor-scale full suspension culture process for recombinant baculovirus rBac-V1-V2-F2-σC In this example, the recombinant baculovirus rBac-V1-V2-F2-σC was cultured in a 1000 L bioreactor using fully suspended High5 cells. The steps are as follows (see the schematic diagram of the process). Figure 8 ): 1. Use triangular ventilated shake flask to culture 200 mL of fully suspended High5 cells. The culture medium used is fully suspended serum-free culture medium, and the culture conditions are as follows: 27.0℃, culture time is 48h; 2. For the fully suspended High5 cells obtained in step 1, the initial density was 8.0×10 5 cells / mL, and the total volume was 1000 mL in a shake flask. The medium used was a full suspension serum-free medium. The culture conditions were as follows: 27.0°C, and the culture time was 48 h; 3. Use triangular shake flasks to pass recombinant baculovirus rBac-V1-V2-F2-σC with full suspension cells High5 cells and prepare seed virus: the culture medium used is full suspension serum-free culture medium, the culture conditions are as follows: 27.0℃, culture time is 96-120h, and the prepared virus titer is 10 -7.5 / 0.1mL, IBDV agar titer ≥1:32, FADV agar titer ≥1:32; 4. 50L bioreactor (working volume 30L) High5 healthy full suspension cell culture: The full suspension High5 cells obtained in step 2 were cultured at an initial density of 8.0×10 5 cells / mL, and the total volume of the shake flask was 6000 mL for further expansion. After 48 h of culture, the cells were inoculated into a 50 L bioreactor for full suspension culture. The culture medium was a full suspension serum-free medium, and the initial cell density was 100 × 10 4 -150×10 4 cells / ml, the reactor culture parameters were set as follows: temperature 27.5°C, DO: 55%, rotation speed: 70-110rpm, pH 6.1, and cell culture cycle of 48h; 5. 250L bioreactor (working volume 120L) High5 full suspension cell culture: The High5 full suspension cells obtained in step 4 were inoculated into a 250L bioreactor for full suspension culture. The culture medium was a full suspension serum-free culture medium with an initial cell density of 100-150×10 4 cells / ml, the reactor culture parameters were set as follows: temperature 27.5°C, DO: 55%, rotation speed: 70-90rpm, pH 6.1, and cell culture cycle of 48h; 6. 1000 L bioreactor (initial working volume 450 L) High5 full suspension cells inoculated with recombinant baculovirus rBac-V1-V2-F2-σC culture: The High5 full suspension cells obtained in step 5 were inoculated into a 1000 L bioreactor. The culture medium was a full suspension serum-free culture medium. The initial cell inoculation density was 100 × 10 4 cells / ml, and the cells were cultured for 48 h. When the cell density reached 5.0-6.0×10 6cells / ml, inoculate the recombinant baculovirus rBac-V1-V2-F2-σC prepared in step 3, with an MOI of about 0.5. When inoculating the virus, add a full suspension serum-free medium equal to the culture medium, and dilute the cell number to 2.8×10 6 -3.0×10 6 cells / ml. The reactor culture parameters were set as follows: temperature: 27.5°C, DO: 55%, rotation speed: 80 rpm, pH 6; 7. Take samples every few hours for cell density and viability analysis (degree of cytopathic effect). When the cell viability drops below 70% and the cell density drops to 1.5×10 6 When the concentration of the virus liquid is less than 1:1 cells / mL, the culture is terminated and the virus liquid is harvested (900 L). The virus liquid is tested for titer by agar amplification, and the results are as follows: IBDV ≥ 1:32, FADV ≥ 1:32.

[0060] The virus solution was inactivated at 37°C (with formaldehyde solution) for 24 hours to obtain the virus antigen. The antigen was centrifuged and emulsified to prepare the vaccine for animal immunization tests.

[0061] The morphology of healthy cells in full suspension High5 cultured for 48 hours is shown under the microscope. Figure 7 The left picture shows the microscopic morphology of the whole suspension High5 healthy cells inoculated with the recombinant baculovirus rBac-V1-V2-F2-σC and cultured for 48 hours. Figure 7 The main index monitoring results of the culture process of High5 full suspension cells are shown in Tables 2 to 4. The growth and proliferation trend curves of High5 healthy cells in 50L, 250L, and 1000L bioreactors (XF-High5-50L, XF-High5-250L, and XF-High5-1000L, respectively) are shown in Figure 2. Fig. 9 The morphology of healthy High5 cells cultured in a 1000 L bioreactor for 72 hours is shown under the microscope. Fig.10 Left: The microscopic morphology of the 1000 L bioreactor fully suspended High5 healthy cells inoculated with the recombinant baculovirus rBac-V1-V2-F2-σC after 72 hours of culture. Fig.10 The right picture shows the titer of FADV (left) and IBDV (right) antigen agar amplification in the rBac-V1-V2-F2-σC virus fluid harvested from the 1000 L bioreactor. Fig.11 .

[0062] Table 2 50L bioreactor High5 full suspension cell culture Table 3 250L bioreactor High5 full suspension cell culture Table 4 1000L bioreactor High5 full suspension cell culture and culture after virus inoculation Example 5 In this example, the recombinant baculovirus rBac-V1-V2-F2-σC strain virus liquid obtained by full suspension culture in Example 4 was prepared into a quadruple bivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal virus (classical strain + variant strain) + group I type 4 avian adenovirus + avian reovirus. Different batches of recombinant baculovirus rBac-V1-V2-F2-σC strain virus liquid were used for three preparations. The specific method is as follows: 1. Vaccine preparation (1) Preparation of oil phase: Take 95 parts of mineral oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tube and heat them to 80°C, then add 5 parts of Siben-80, keep the temperature at 115°C for 30 minutes, and cool to complete the oil phase preparation.

[0063] (2) Preparation of aqueous phase: Take 95 parts of inactivated H9 subtype avian influenza virus F strain, 90 parts of recombinant baculovirus rBac-V1-V2-F2-σC strain culture medium, and 5 parts of sterilized Tween-80, mix thoroughly to completely dissolve the Tween-80.

[0064] (3) Emulsification: Put 2 portions of the oil phase into an emulsification tank, start the motor, rotate and stir slowly, and slowly add 1 portion of the water phase. Then stir at 3500 r / min for 5 minutes and 10000 r / min for 5 minutes to complete the emulsification preparation. After emulsification, take 10 ml of the vaccine and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes. The amount of water precipitated at the bottom of the tube should not exceed 0.5 mL.

[0065] 2. Vaccine properties Appearance: Milky white uniform emulsion.

[0066] Dosage form: Oil-in-water type. Take a clean pipette and draw a small amount of vaccine and drop it on the surface of clean cold water. Except for the first drop, it will not spread.

[0067] Stability: Pipette 10 mL of the vaccine into a centrifuge tube and centrifuge at 3000 rpm for 15 minutes. The aqueous phase precipitated at the bottom of the tube should not exceed 0.5 mL.

[0068] 3. Sterility test The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results showed that there was no sterile growth.

[0069] Example 6 Safety test of quadruple bivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group I type 4 avian adenovirus + avian reovirus The safety test of the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classical strain + variant strain) + group I type 4 avian adenovirus + avian reovirus prepared in Example 5 was carried out. The specific method is as follows: 40 14-day-old SPF chickens were divided into 4 groups, each with 10 chickens. Three groups of H9 subtype avian influenza virus + avian bursal virus (classic strain + variant strain) + group Ⅰ type 4 avian adenovirus + avian reovirus quadruple inactivated vaccine (batch numbers 2023001, 2023002, and 2023003) prepared in three batches were subcutaneously injected at a dose of 2 mL / chicken; 10 SFP chickens in the control group were subcutaneously injected with sterile saline at a dose of 2 mL / chicken. Chickens in each group were raised and managed under the same conditions and observed for 14 consecutive days; if there was death, the dead chickens were dissected one by one to observe whether the internal organs had lesions; and the live chickens were observed for adverse reactions. All surviving chickens were killed 14 days after vaccination to observe whether the internal organs had lesions. The local inflammation and tissue lesions of the chickens were judged. The test results are shown in Table 5.

[0070] Table 5 Safety test results The above results indicate that the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classical strain + variant strain) + group I type 4 avian adenovirus + avian reovirus prepared by the present invention has high safety.

[0071] Example 7 Immune efficacy test of quadruple bivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group I type 4 avian adenovirus + avian reovirus The immune efficacy of the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classical strain + variant strain) + group I type 4 avian adenovirus + avian reovirus prepared in Example 5 was tested, and the specific method is as follows: Take 60 SPF chickens of 3 weeks old and divide them into 6 groups, wherein the first to third groups are three batches of H9 subtype avian influenza virus + avian bursal virus (classical strain + variant strain) + I group 4 type avian adenovirus + avian reovirus quadruple bivalent inactivated vaccine (full suspension cell culture, batch number 2020001, 2020002, 2020003) of Example 5, the fourth group is an inactivated vaccine prepared by IBDV whole virus antigen, FADV whole virus antigen and ARV whole virus antigen cultured by adherent cells (available from Qianyuanhao Biological Co., Ltd.), and the fifth group is a control group, each group of 10 SPF chickens. Each was injected intramuscularly in the chest, 0.3 ml each. Blood was collected for 21 days to detect HI antibodies, and the test was repeated three times. The test results are shown in Tables 6-8.

[0072] Table 6 Results of the first test of the immune efficacy of the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + avian adenovirus type 4 group I + avian reovirus Table 7 The results of the second test of the immune efficacy of the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group Ⅰ type 4 avian adenovirus + avian reovirus Table 8 The results of the third test of the immune efficacy of the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group Ⅰ type 4 avian adenovirus + avian reovirus The above results show that when chickens (0.3 ml / bird) were immunized with the three groups of H9 subtype avian influenza virus + avian bursal virus (classic strain + variant strain) + group Ⅰ type 4 avian adenovirus + avian reovirus quadruple bivalent inactivated vaccine, the serum IBDV agar titer was ≥1:8 and the serum FADV agar titer was ≥1:8 21 days after immunization, and the serum ELISA test results were all positive. Among them, the antibody titer induced by IBDV was better than that of the vaccine prepared by the whole IBDV virus cultured in adherent cells. Since the antibodies produced by the vaccine prepared by the whole virus cultured in FADV adherent cells cannot be detected by the agar gel diffusion test method for the time being, the subsequent test will be continued using the virus attack method.

[0073] Example 8 Challenge test of quadruple bivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group I type 4 avian adenovirus + avian reovirus 1. Use virulent strains of avian bursal disease virus and avian adenovirus, respectively, and conduct the test using the mucosal challenge method.

[0074] 2. Virus attack experiment The strong toxins of IBDV (BC 6 / 85 strain), IBDV super strain (IBDV-ZZ-HB strain), IBDV variant strain (IBDV-ZZ-SQ strain) and FADV (FADV-ZZ-LS strain) were used to challenge SPF chickens 21 days after immunization with the quadrivalent inactivated vaccine of H9 subtype avian influenza virus + avian bursal disease virus (classic strain + variant strain) + group I type 4 avian adenovirus + avian reovirus. A challenge control group was set up with 5 chickens in each group.

[0075] Infectious bursal disease virus BC 6 / 85 strain (CVCC AV7) was identified, stored and supplied by the China Veterinary Drug Administration. The virulent bursal disease virus strain IBDV-ZZ-HB, the variant bursal disease virus strain IBDV-ZZ-SQ and the virulent group Ⅰ type 4 avian adenovirus strain FADV-ZZ-LS were isolated by our laboratory.

[0076] 2.1 Attack Methods The classic strain of bursal bursal virus BC 6 / 85, the super strong strain of IBDV (IBDV-ZZ-HB strain), and the variant strain of IBDV (IBDV-ZZ-SQ strain) were diluted to 100 BID / 0.1ml with sterile saline, and the eye drop + cloaca challenge was performed, with a challenge dose of 0.2ml / bird. The strong strain of group I type 4 fowl adenovirus (FADV-ZZ-LS strain) was diluted to 2×10 7.0 TCID50 / 0.1ml, subcutaneous injection of the neck was used for the challenge, and the challenge dose was 0.2ml.

[0077] The test groups are as follows: the first to third groups are three batches of H9 subtype avian influenza virus + avian bursal virus (classical strain + variant strain) + group Ⅰ type 4 avian adenovirus + avian reovirus quadrivalent inactivated vaccine (batch numbers 2020001, 2020002, 2020003) in Example 5, the fourth group is an inactivated vaccine prepared with IBDV whole virus antigen and FADV whole virus antigen cultured in adherent cells, and the fifth group is the control group. After 21 days of immunization, the classical strain BC 6 / 85 of avian bursal virus, the super strain of IBDV (IBDV-ZZ-HB strain), the variant strain of IBDV (IBDV-ZZ-SQ strain) and the strong strain of group Ⅰ type 4 avian adenovirus FADV-ZZ-LS were used for attack, with 5 chickens in each attack group. The observation results are shown in Table 9. Representative anatomical diagrams of the diseased and dead chickens in the control group of the IBDV attack test are shown in Fig.12 The chickens showed bleeding spots at the junction of the glandular stomach and gizzard, enlarged cysts of Fabricius, and internal bleeding. The representative anatomical diagram of the dead chickens in the control group of the FADV challenge test is shown in Fig.13 The chickens developed enlarged, yellow and brittle livers, posterior displacement of the stomach, pericardial effusion with yellowish jelly-like lesions near the heart, and green stools.

[0078] Table 9 Observation results of the challenge experiment 2.2 Attack results At 120 hours after infection, the surviving chickens in the avian bursal virus infection group were killed, the bursa of Fabricius was ground and filtered, and inoculated into adherent DF-1 cells in good condition after passing the sterility test; the surviving chickens in the group I type 4 avian adenovirus group had their livers ground and filtered, and inoculated into adherent LMH cells in good condition after passing the sterility test, and cultured them statically for 96 hours to observe their pathological conditions.

[0079] The virus isolation results after challenge and challenge determination are shown in Table 10.

[0080] Table 10 Attack results Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a recombinant vector, characterized in that: pFastBac Dual was used as a vector, and the VP2 gene of the classical strain of avian bursal disease virus was inserted between the restriction sites of EcoRI and XbaI restriction endonucleases of the vector, thereby obtaining a recombinant plasmid vector into which the VP2 gene of the classical strain of avian bursal disease virus was inserted, which was named pDual-V1; The VP2 gene of the variant strain of avian bursal disease virus and the pDual-V1 were double-digested with XhoI and KpnI, respectively, and then ligated by enzymes to obtain a recombinant plasmid vector into which the VP2 gene of the classical strain of avian bursal disease virus and the VP2 gene of the variant strain of avian bursal disease virus were simultaneously inserted, and was named pDual-V1-V2; Inserting the Fiber-2 gene of group I type 4 fowl adenovirus into the EcoRI and XbaI restriction sites of the vector to obtain a recombinant plasmid vector with the Fiber-2 gene of group I type 4 fowl adenovirus inserted therein, named pDual-F2; The avian reovirus σC gene sequence and the pDual-F2 were double-digested with XhoI and KpnI, respectively, and then ligated to obtain a recombinant plasmid vector into which both the group I type 4 avian adenovirus fiber-2 gene and the avian reovirus σC gene sequence were inserted, and the vector was named pDual-F2-σC. The pDual-V1-V2 is double-digested with PmeI / AvrII to recover small fragments; the pDual-F2-σC is double-digested with SpeI / NruI to recover large fragments, and then mixed enzyme ligation is performed to obtain a recombinant plasmid vector containing the VP2 gene of the classical strain of avian bursal disease virus, the VP2 gene of the variant strain of avian bursal disease virus, the Fiber-2 gene of the group I type 4 avian adenovirus, and the σC gene of the avian reovirus, which is named pDual-V1-V2-F2-σC; The sequence of the VP2 gene of the classical strain of avian bursal disease virus is shown in SEQ ID No. 1; The sequence of the VP2 gene of the avian bursal disease virus variant is shown in SEQ ID No. 2; The sequence of the Fiber-2 gene of the group I type 4 avian adenovirus is shown in SEQ ID No. 3; The sequence of the avian reovirus σC gene is shown in SEQ ID No.4; Preferably, the primer sequences used to amplify the VP2 gene of the classical strain of avian bursal virus, the VP2 gene of the variant strain of avian bursal virus, the Fiber-2 gene of the group I type 4 avian adenovirus and the σC gene of the avian reovirus are as shown in SEQ ID No.5-12.

2. A method for preparing a recombinant baculovirus, characterized in that: include: (1) Clone the DNA molecule into Escherichia coli DH10 Bac competent cells and screen and identify the recombinant baculovirus Bacmid; The DNA molecule comprises a sequentially connected Fiber-2 gene of group I type 4 avian adenovirus, a VP2 gene of a variant strain of avian bursal virus, a VP2 gene of a classical strain of avian bursal virus, and a σC gene of an avian reovirus; the sequences of the Fiber-2 gene of group I type 4 avian adenovirus, a VP2 gene of a variant strain of avian bursal virus, a VP2 gene of a classical strain of avian bursal virus, and a σC gene of an avian reovirus are as described in claim 1; (2) transfecting SF9 cells with the recombinant baculovirus Bacmid obtained in step (1), and harvesting the recombinant baculovirus after culture; Preferably, in step (1), the recombinant vector prepared by the method described in claim 1 is used to transform Escherichia coli DH10 Bac competent cells, so as to clone the DNA molecule into the Escherichia coli DH10 Bac competent cells.

3. A recombinant baculovirus, characterized in that Prepared by the method of claim 2; or, the deposit number of the recombinant baculovirus is CCTCC NO: V202448.

4. A DNA molecule, characterized in that As described in claim 2.

5. A DNA molecule, characterized in that The nucleotide sequence is shown as SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.

4.

6. A biological material containing the DNA molecule according to claim 4 or 5, wherein the biological material is an expression cassette, a recombinant vector, a transgenic cell line, a recombinant bacterium or a recombinant virus.

7. A method for preparing a virus culture solution of the recombinant baculovirus according to claim 3, characterized in that: include: (1) Cultivating whole suspension cells; preferably, the whole suspension cells are insect cells, cultured in a whole suspension serum-free medium, and more preferably, the insect cells are High5 cells; (2) inoculating the recombinant baculovirus according to claim 3 for suspension culture; (3) Harvesting virus culture fluid; Preferably, in step (2), when the recombinant baculovirus according to claim 3 is inoculated, the growth density of the whole suspension cells is 5.0×10 6 -6.0×10 6 cells / mL, add medium when inoculating the virus, and dilute the whole suspension cell number to 2.8×10 6 -3.0×10 6 cells / mL; and / or, the virus inoculation amount is MOI=0.5-2, and the culture conditions are: temperature 27±1℃, dissolved oxygen 55-65%, stirring speed 80 rpm, pH 6.0-6.2; Preferably, in step (3), when the viability of the whole suspension cells drops below 70% and the density drops to 2×10 6 When the concentration was below 10 cells / mL, the culture was terminated, and the whole suspended cells were disrupted and the virus culture fluid was collected.

8. A virus culture solution, characterized in that Prepared by the method described in claim 7.

9. Use of the method for preparing a recombinant vector according to claim 1, the method for preparing a recombinant baculovirus according to claim 2, the recombinant baculovirus according to claim 3, the DNA molecule according to claim 4 or 5, the biological material according to claim 6, the method for preparing a virus culture solution of the recombinant baculovirus according to claim 3, or the virus culture solution according to claim 8 in preparing a vaccine.

10. A vaccine, characterized in that Comprising the virus culture solution of claim 8.

Images