Modified gosling plague virus VP3 gene and application thereof

Abstract

The invention provides a modified gosling plague virus VP3 gene and application thereof. The nucleotide sequence of the modified gosling plague virus VP3 gene is shown as SEQ ID NO: 1. The expression quantity of the VP3 protein is improved by optimizing and modifying a VP3 gene sequence and adding a translation enhancement sequence, so that the efficient soluble expression of the VP3 protein is realized. The method comprises the following steps: constructing a recombinant expression vector by using a modified gosling plague virus VP3 gene, converting recombinant expression bacteria containing expression chaperonin by using the recombinant expression vector, and co-expressing recombinant escherichia coli of VP3 protein and chaperonin; the recombinant expression bacteria are subjected to induced expression, supernatant is purified to obtain VP3 protein, and the vaccine prepared by mixing and emulsifying the VP3 protein and an adjuvant has good immunogenicity and stability.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering of poultry biological products, and in particular to a modified goose plague virus VP3 gene and application thereof. Background Art

[0002] Gosling Plague (GP), also known as goose parvovirus disease, is an acute or subacute septicemic infectious disease of goslings caused by goose parvovirus (GPV). The disease mainly affects goslings and Muscovy ducklings aged 4-20 days, and is characterized by acute enteritis and lesions of solid organs such as the liver, kidney, and heart. It has the characteristics of rapid transmission, high morbidity and mortality. At present, the disease is still one of the most serious infectious diseases that endanger the goose farming industry. It is prevalent to varying degrees every year, causing considerable economic losses to the goose farming industry.

[0003] GPV is a genome containing two open reading frames (ORFs), which can be divided into left ORF and right ORF. The left ORF encodes non-structural proteins NS1 and NS2; the right ORF encodes structural proteins VP1, VP2, and VP3, and the three structural proteins share the same stop codon. The length of VP1 is 2199bp (87kDa), the length of VP2 is 1764bp (67kDa), and the length of VP3 is 1605bp (58kDa). Therefore, the amino acid sequence of VP3 is contained in VP2, and the complete amino acid sequences of VP2 and VP3 are contained in VP1. This structure is called a "basket structure" (the start codons are different but share the same stop codon). The VP3 protein is the main capsid protein of the virus and is the most conservative, accounting for about 80% of the total protein. It contains the main epitopes of the virus and can induce the body to produce neutralizing antibodies. It is one of the main proteins in subunit vaccine research.

[0004] The preparation of subunit antigens using the E. coli prokaryotic expression system has the characteristics of strong safety, low production cost, and easy preparation and production. Currently, GPV VP3 protein is expressed in the form of inclusion bodies in the E. coli expression system, which limits the application of subunit antigens in vaccine preparation.

[0005] Therefore, the prior art needs to be further developed. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned technical deficiencies and provide a modified goose plague virus VP3 gene and its application to solve the technical problem in the related art that the VP3 gene of goose parvovirus is mainly expressed in the form of insoluble inclusion bodies in the prokaryotic expression system and the expression amount is low.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical scheme: a modified goose plague virus VP3 gene is provided, and the nucleotide sequence is shown in SEQ ID NO:1.

[0008] Furthermore, a translation enhancing sequence is added after the start codon, and the nucleotide sequence is shown in SEQ ID NO:2. An expression vector contains the above-mentioned goose plague virus VP3 gene.

[0009] A host bacterium contains the above expression vector.

[0010] The goose plague virus VP3 protein is obtained by expressing the goose plague virus VP3 gene.

[0011] The use of the above-mentioned goose plague virus VP3 protein in the preparation of goose plague virus VP3 subunit vaccine. A goose plague virus VP3 subunit vaccine contains an antigen made from the goose plague virus VP3 protein.

[0012] The preparation method of the above-mentioned vaccine includes the following steps: inserting the modified VP3 gene of goose plague virus into the multiple cloning site of pET-28a vector to construct a recombinant expression vector; transforming the recombinant expression vector into recombinant expression bacteria containing expression chaperone protein, and recombinant Escherichia coli capable of co-expressing VP3 protein and chaperone protein; collecting the cells of the recombinant expression bacteria after induction of expression, ultrasonically disrupting and obtaining the supernatant, purifying the supernatant to obtain VP3 protein, and mixing the VP3 protein with an adjuvant and emulsifying to prepare a vaccine.

[0013] Furthermore, the recombinant expression bacteria were induced to express overnight at 25°C and 160rpm using lactose and tetracycline as inducers for 12 to 16 hours. The VP3 protein was purified using an 8-12% PEG6000 precipitation method, and 1% Triton-114 was used to remove endotoxins during the purification process to obtain a VP3 protein solution for subunit vaccine preparation.

[0014] Furthermore, the adjuvant is a white oil adjuvant, and the VP3 protein and the white oil adjuvant are fully mixed in a ratio of 1:2 to 1:3 and then emulsified.

[0015] Beneficial effects: The present invention optimizes the VP3 protein gene and adds a translation enhancing sequence after the start codon that can improve the expression of the target protein to increase the expression amount of the VP3 protein. At the same time, the target protein and the Escherichia coli Tig chaperone protein are co-expressed in a host expression bacteria to increase the expression amount of the VP3 protein, thereby achieving efficient soluble expression of the VP3 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the PCR identification diagram of GPV-VP3 suspected disease material; where M is 2000bp Marker; 1 is negative control; 2~8 are GPV identification products; Figure 2 The electrophoresis results of the VP3 gene sequence shown in SEQ ID NO: 1 in Escherichia coli carrying Tf2 and Tf16 chaperone plasmids respectively; Figure 3 The electrophoresis results of the optimized VP3 gene sequence shown in SEQ ID NO: 2 in common Escherichia coli and Escherichia coli carrying Tf2 and Tf16 chaperone plasmids, respectively; Figure 4 This is the gVP3 (DE3 / Tf16) antigen agar amplification titer test chart; Figure 5 This is the gVP3 (DE3 / Tf2) antigen agar amplification titer test chart; Figure 6 This is a graph of the gVP3 antigen agar amplification titer test in high-density fermentation. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0018] Example 1 1. Obtaining the target gene for expression GPV infected with goose plague virus was amplified and identified from the suspected goose plague virus diseased material sent for inspection from a farm. Viral DNA was extracted from the diseased material using a viral DNA extraction kit, and the VP3 gene was amplified using a PCR amplification kit. The VP3 gene fragment was recovered by gel, connected to the pMD-19T cloning vector, and transformed into the top10 competent Escherichia coli. The single clone colony was selected and sent to Shanghai Bioengineering Co., Ltd. for sequencing to obtain the VP3 gene sequence and perform sequence analysis.

[0019] Amplification primer sequences: GPV-VP3-F:5'-ATGGCAGAGGGAGGAAGC-3' GPV-VP3-R:5'-TTACAGATTCTGAGTCAAATATCTG-3' Specific steps: (1) Viral DNA extraction (the kit is Omega's Viral DNA Kit). The operation steps refer to the kit manual.

[0020] (2) Amplification of VP3 gene: GPV-VP3 identification F / R primers were used to perform PCR amplification on the diseased sample DNA. The PCR system and procedure were as follows: 2×FS PCR Master Mix 25ul GPV-VP3 F 2ul GPV-VP3 R 2ul DNA template 3ul RNase free water 18ul Total 50ul 98℃ 10s; 98℃ 5s-55℃ 5s-72℃ 20s (34 cycles); 72℃ 5min.

[0021] After the target fragment was recovered from the gel, it was connected to the pMD-19T cloning vector and transformed into top10 competent E. coli after overnight connection at 4°C. A single clone colony was selected and sent to Shanghai Bioengineering Co., Ltd. for sequencing and identification. The analysis confirmed that the strain was GPV (such as Figure 1 ), the original sequence of GPV-VP3 is shown in SEQ ID NO:3.

[0022] 2. Construction of recombinant expression vector The VP3 gene sequence was designed and optimized according to the codon preference of Escherichia coli and sent to Shanghai Shenggong Biotechnology Co., Ltd. for gene synthesis to obtain cloning plasmids with the optimized VP2 gene sequence, namely pET-28a-VP3 (only the codon sequence was optimized, the sequence is shown in SEQ ID NO: 1) and pET-28a-gVP3 (codon sequence optimization and addition of translation enhancement sequence, the sequence is shown in SEQ ID NO: 2), and the translation enhancement sequence is CTGACCCTG.

[0023] 3. Preparation of recombinant expression bacteria and expression identification (1) Preparation of recombinant expression bacteria The pET-28a-VP3 recombinant plasmid was transformed into Ecoli.BL21 (DE3) competent cells (with pTf16 chaperone plasmid) and Ecoli.BL21 (DE3) competent cells (with pG-Tf2 chaperone plasmid), respectively, and the positive recombinant bacteria pET-28a-VP3 (DE3 / Tf16) and pET-28a-VP3 (DE3 / Tf2) were screened and identified.

[0024] The pET-28a-gVP3 recombinant plasmid was transformed into Ecoli.BL21 (DE3) competent cells, Ecoli.BL21 (DE3) competent cells (with pTf16 chaperone plasmid) and Ecoli.BL21 (DE3) competent cells (with pG-Tf2 chaperone plasmid), and the positive recombinant bacteria pET-28a-gVP3 (DE3), pET-28a-gVP3 (DE3 / Tf16) and pET-28a-gVP3 (DE3 / Tf2) were screened and identified.

[0025] (2) Laboratory expression verification Bacterial strain recovery: Inoculate the positively expressed bacteria into 5 ml TB liquid culture medium containing chloramphenicol and kanamycin resistance (20 ml test tube) at a ratio of 1%, and culture overnight at 37°C 220 rpm (14-16 h).

[0026] Induce expression: Take the expression bacteria cultured overnight and inoculate them into 40 ml TB liquid culture medium containing chloramphenicol and kanamycin resistance (250 ml triangle bottle) at a ratio of 1%, culture at 37℃ 220rpm for 2~3h, and monitor the OD of the bacterial solution 600 , when OD 600 When the concentration reaches about 0.8, take out the flask and place it in a 25℃ water bath to cool for 20~30min. Add inducer lactose (final concentration 1g / L) and tetracycline (final concentration 1~5ng / ml), culture overnight (14~16h) at 25℃ and 160rpm, and collect the bacteria by centrifugation. Weigh the collected bacteria, resuspend the bacteria with PBS at a mass volume ratio of 1:20, add 20mg / ml lysozyme at a ratio of 1:1000, freeze and thaw once, and then ultrasonically break it. Centrifuge at 12000rpm for 10min to obtain the expression supernatant and precipitate, resuspend the precipitate with PBS and set aside.

[0027] SDS-PAGE electrophoresis expression identification: After adding 4× protein electrophoresis loading buffer to the supernatant and precipitate after ultrasonic disruption, boil them in boiling water for 7-10 minutes. Take 10 μL of each sample and load it on polyacrylamide gel electrophoresis. The electrophoresis results are shown in Figure 2 and Figure 3 , Figure 2 The expression results of the VP3 gene sequence shown in SEQ ID NO:1 in Escherichia coli carrying Tf2 and Tf16 chaperone plasmids respectively (the red arrow indicates the chaperone protein, and the black arrow indicates the VP3 protein), wherein 1 is the total protein of VP3 (DE3 / Tf2); 2 is the precipitate of VP3 (DE3 / Tf2); 3 is the supernatant of VP3 (DE3 / Tf2); 4 is the total protein of VP3 (DE3 / Tf16); 5 is the precipitate of VP3 (DE3 / Tf16); 6 is the supernatant of VP3 (DE3 / Tf16); Figure 3 These are the expression results of the optimized VP3 gene sequence shown in SEQ ID NO:2 with a translation enhancing sequence in ordinary Escherichia coli and Escherichia coli with Tf2 and Tf16 chaperone plasmids, respectively (the red arrow indicates the chaperone protein and the black arrow indicates the VP3 protein), wherein 1 is the total protein of gVP3 (DE3); 2 is the precipitate of gVP3 (DE3); 3 is the supernatant of gVP3 (DE3 / Tf16); 4 is the total protein of gVP3 (DE3 / Tf2); 5 is the precipitate of gVP3 (DE3 / Tf2); 6 is the supernatant of gVP3 (DE3 / Tf2); 7 is the total protein of gVP3 (DE3 / Tf16); 8 is the precipitate of gVP3 (DE3 / Tf16); 9 and 10 are both the supernatant of gVP3 (DE3).

[0028] (3) Agar immunodiffusion test to detect antigen titer The titer of the supernatant after ultrasonic disruption was tested using the standard positive serum of goose plague (for agar amplification test) from the China Veterinary Drug Administration. The protein supernatant was diluted 2-fold with PBS buffer from 2 1 Dilute to 2 11 . Use an agar puncher to punch holes in the prepared agar plate, add standard positive serum to the center hole, and add 2-fold diluted antigen supernatant to the peripheral holes. After the sample is added, place it in a wet box in a 37°C incubator and observe the test results within 48 hours. When a white precipitate line appears between the center hole and the peripheral holes, and the white precipitate lines are cross-connected, the highest dilution with precipitate lines is determined to be the antigen titer of the VP2 protein. The agar titer of the gVP3 (DE3 / Tf16) antigen expressed in a laboratory shake flask is 1:2 6 The agar titer of gVP3 (DE3 / Tf2) antigen is 1:2 8 The results of the potency of Qiongkuang are shown in Figure 4 and Figure 5 .

[0029] 4. High-density fermentation verification of GPV-gVP3 (DE3 / Tf2) protein Bacterial strain recovery: streak the preserved expression bacteria pET-28a-gVP3 (DE3 / Tf2) on an LB plate with kanamycin and chloramphenicol resistance to pick up a single clone colony or inoculate the frozen bacteria into 5 ml TB liquid medium (20 ml test tube) containing kanamycin and chloramphenicol resistance at a ratio of 1%, and culture at 37°C, 220 rpm overnight (14-16 hours). Use the revived bacterial solution to inoculate the frozen bacteria into 100 ml TB liquid medium (triangular flask) containing kanamycin and chloramphenicol resistance at a ratio of 1%, and culture at 37°C, 220 rpm overnight (14-16 hours).

[0030] High-density fermentation expression: Prepare the culture medium in the fermenter (accounting for 50% of the volume of the fermenter), sterilize and set aside. Before inoculation, stabilize the culture medium temperature in the fermenter at about 37°C, then aseptically inoculate the overnight cultured pET-28a-gVP3 (DE3 / Tf2) expression bacteria into the fermenter at a ratio of 2%, and add the prepared kanamycin (50mg / ml) and chloramphenicol solution (20mg / ml) at a ratio of 1:1000. Cultivate at 37°C for about 6~8h, monitor the wet bacterial weight of the fermentation broth regularly during this period, and start cooling the culture medium when the bacterial weight reaches about 20~30g / L. After about 20min, the tank temperature will stabilize from 37°C to 25°C, then add lactose and tetracycline inducers, and control the temperature at 25°C, pH at 6.80, and dissolved oxygen (DO) at about 20-30% during the induction expression process. Starting from 3 hours after induction, about 10 ml of fermentation liquid was released from the fermenter every 2 hours, and the bacteria were collected by centrifugation and the wet bacterial weight was measured until all the fermentation liquid was released and the bacteria were collected at 14 hours of induction. The final harvested bacterial weight exceeded 100 g / L. The bacteria were resuspended at 1:10 (mass-to-volume ratio) and then crushed with a high-pressure homogenizer. After high-speed centrifugation, the supernatant was taken for detection.

[0031] Agar immunodiffusion test to detect the titer of gVP3 antigen: the method is the same as above, and the titer of gVP3 antigen in the supernatant reaches 1:2 after large-scale fermentation expression in the fermenter 9 The above results show the potency of Qiongkuang. Figure 6 .

[0032] 5. Crude purification of gVP3 protein Take 500ml of the supernatant after crushing, add 28g of solid ammonium sulfate while stirring at 4℃ to make the concentration of ammonium sulfate in the supernatant reach 10%, and add TritonX-114 with a final concentration of 1%. After mixing, let it stand at 4℃ for about 3.5~4h, centrifuge at 10000rpm for 20min, discard the precipitate, retain the supernatant, add 125g of solid ammonium sulfate to the supernatant to a final concentration of 50%, add while stirring, and let it stand at 4℃ overnight after mixing. Centrifuge at 10000rpm for 20min, discard the supernatant, and collect the precipitate. Resuspend the precipitate with the same volume of saline or PBS. After the precipitate is completely re-dissolved, centrifuge at 10000rpm for 20min, discard the precipitate, and keep the supernatant. The purified antigen supernatant was used for agar expansion detection. The results showed that the antigen titer was consistent with that before purification, and if the amount of saline or PBS was reduced when re-dissolving the precipitate, the protein concentration could be achieved and the antigen titer could be increased. In actual application, the dosage of the reconstituted solution can be appropriately adjusted according to the potency of the desired seedlings.

[0033] 6. Preparation and application of GPV-gVP3 subunit vaccine (1) Vaccine preparation The purified gVP3 antigen solution was diluted with physiological saline to an agar titer of 1:32, and emulsified with the prepared white oil adjuvant at a ratio of 1:2~1:3 using an IKA disperser, and 3‰ of 40% formaldehyde solution was added to prepare the GPV-gVP3 genetically engineered subunit vaccine.

[0034] (2) Vaccine efficacy test evaluation Thirty one-week-old goslings were purchased and randomly divided into three groups, with 10 in each group. The immunization injection method was multiple subcutaneous injections at the back of the neck. Group 1 was the immunization and challenge group, with 0.2 ml / goose injected; Group 2 was the blank control group (no immunization and no challenge); Group 3 was the challenge control group (no immunization but challenge).

[0035] Antibody detection: Blood was collected at 0d / 7d / 14d to measure the titer of agar-amplified antibodies. At 0d, the titer of serum antibodies in all goslings was 0. At 7d, the titer of serum in the control group was 0, and 6 sera in the immunized group were positive, with a titer greater than 1:8. At 14d, the antibody positivity rate in the immunized group was 100%, of which 2 had a titer greater than 1:32, and the rest were greater than 1:16.

[0036] Virus shedding test after challenge: The virus was challenged 14 days after immunization, and oral and anal swabs were collected for antigen detection on days 3, 5, 7, and 14 after challenge. One gosling in the immunized challenge group 1 excreted virus on day 3, and the rest did not excrete virus, and no excretion was detected later; all goslings in the blank control group in group 2 were negative; all goslings in the challenge control group in group 3 began to excrete virus on the third day, and were still excreting virus until the 14th day test.

[0037] The above animal immune efficiency evaluation test proves that the subunit antigen prepared by the present invention can be used for the preparation and use of goose plague virus VP3 protein subunit vaccine.

[0038] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0039] Optionally, the specific examples in this embodiment may refer to the examples described in the above embodiments, and this embodiment will not be described in detail here.

[0040] The serial numbers of the above-mentioned embodiments of the present application are for description only and do not represent the advantages or disadvantages of the embodiments.

[0041] In the above embodiments of the present application, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, please refer to the relevant description of other embodiments.

[0042] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A modified goose plague virus VP3 gene, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

1.

2. The goose plague virus VP3 gene according to claim 1, characterized in that: A translation enhancing sequence is also added after the start codon, and the nucleotide sequence is shown in SEQ ID NO:

2.

3. An expression vector, characterized in that: Contains the goose plague virus VP3 gene according to claim 1 or 2.

4. A host bacterium, characterized in that Contains the expression vector according to claim 3.

5. Goose plague virus VP3 protein, characterized in that Obtained by expressing the goose plague virus VP3 gene according to claim 1 or 2.

6. Use of the goose plague virus VP3 protein according to claim 5 in the preparation of a goose plague virus VP3 subunit vaccine.

7. A goose plague virus VP3 subunit vaccine, characterized in that: An antigen prepared from the goose plague virus VP3 protein as described in claim 5.

8. The method for preparing the vaccine according to claim 7, characterized in that: The method comprises the following steps: inserting the modified VP3 gene of goose plague virus into the multiple cloning site of pET-28a vector to construct a recombinant expression vector; transforming the recombinant expression vector into recombinant expression bacteria containing expression chaperone protein, and recombinant Escherichia coli capable of co-expressing VP3 protein and chaperone protein; collecting the bacterial cells of the recombinant expression bacteria after induction of expression, performing ultrasonic crushing and obtaining supernatant, purifying the supernatant to obtain VP3 protein, and mixing the VP3 protein with an adjuvant and emulsifying to prepare a vaccine.

9. The preparation method according to claim 8, characterized in that: The recombinant expression bacteria were induced to express overnight at 25°C and 160rpm using lactose and tetracycline as inducers for 12 to 16 hours. The VP3 protein was purified using 8-12% PEG6000 precipitation method. During the purification process, 1% Triton-114 was used to remove endotoxins to obtain the VP3 protein solution for subunit vaccine preparation.

10. The preparation method according to claim 8, characterized in that: The adjuvant is a white oil adjuvant, and the VP3 protein and the white oil adjuvant are fully mixed in a ratio of 1:2 to 1:3 and then emulsified.

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