Avian pathogenic escherichia coli-Newcastle disease virus chimeric protein, recombinant plasmid and application

Abstract

The invention discloses an avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein, a recombinant plasmid and application, and the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is obtained by recombinant expression of a coding gene of avian pathogenic Escherichia coli outer membrane protein A and a coding gene of Newcastle disease virus F protein. The recombinant plasmid comprises a vector and a target gene, and the target gene comprises a coding gene of avian pathogenic escherichia coli outer membrane protein A and a coding gene of Newcastle disease virus F protein. The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is applied to preparation of avian pathogenic Escherichia coli vaccines and / or Newcastle disease virus vaccines. The coding genes of the avian pathogenic Escherichia coli outer membrane protein A and the Newcastle disease virus F protein are subjected to recombination and induced expression to obtain the chimeric protein with immunogenicity similar to that of the avian pathogenic Escherichia coli outer membrane protein A and that of the Newcastle disease virus F protein, and a direction is provided for development of APEC and NDV novel duplex vaccines.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein, a recombinant plasmid and applications. Background Art

[0002] Avian Pathogenic Escherichia coli (APEC) is an extraintestinal pathogenic Escherichia coli that can cause various local and systemic infections in chickens, turkeys, ducks and many other poultry, leading to air sac inflammation, sepsis, omphalitis, salpingitis, peritonitis and E. coli granulomas, etc. It is one of the main causes of poultry mortality and morbidity, and seriously endangers the development of the poultry industry. APEC serotypes are numerous and complex, and there is a lack of effective cross-immunity protection between serotypes, which brings certain difficulties to the vaccine prevention and treatment of APEC.

[0003] Newcastle Disease (ND) is an acute, highly contagious disease caused by a virulent strain of the Newcastle Disease virus, mainly infecting poultry. Although existing vaccines have reduced the morbidity and mortality of Newcastle Disease, they cannot provide complete protection, and immune failure and immune virus infection persist for a long time.

[0004] Since there are no reports on the research of avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein at home and abroad, it is an urgent problem to be solved by the present invention to provide a chimeric protein that can achieve joint prevention and control of the above two diseases. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to recombine and induce the expression of the coding genes of avian pathogenic Escherichia coli outer membrane protein A and Newcastle disease virus F protein, obtain a chimeric protein with immunogenicity similar to that of avian pathogenic Escherichia coli outer membrane protein A and Newcastle disease virus F protein, achieve dual specificity for recognizing avian pathogenic Escherichia coli and Newcastle disease virus, and provide a direction for the development of new APEC and NDV dual vaccines.

[0006] In order to achieve the above object, the present invention provides an avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein, which is obtained by inducing expression of a gene encoding an outer membrane protein A of avian pathogenic Escherichia coli and a gene encoding a Newcastle disease virus F protein after recombination.

[0007] Preferably, the amino acid sequence of the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is as shown in SEQ ID No: 1.

[0008] The present invention also provides a recombinant plasmid for expressing the above-mentioned avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein, wherein the recombinant plasmid comprises a vector and a target gene, and the target gene comprises a gene encoding an outer membrane protein A of avian pathogenic Escherichia coli and a gene encoding a Newcastle disease virus F protein.

[0009] Preferably, the target gene is obtained by embedding the third coding gene fragment of the longest extramembrane region of Newcastle disease virus F protein between the first coding gene fragment of the N-terminal part of the extramembrane region of avian pathogenic Escherichia coli outer membrane protein A and the second coding gene fragment of the C-terminal part of the extramembrane region of avian pathogenic Escherichia coli outer membrane protein A.

[0010] Preferably, the length of the first coding gene fragment is 684 bp;

[0011] And / or, the length of the second coding gene fragment is 294 bp;

[0012] And / or, the length of the third coding gene fragment is 1089 bp.

[0013] Preferably, the nucleotide sequence of the target gene is as shown in SEQ ID No: 2.

[0014] Preferably, the primers used in the construction of the recombinant plasmid include a first primer pair as shown in SEQ ID No: 3 and SEQ ID No: 4, a second primer pair as shown in SEQ ID No: 5 and SEQ ID No: 6, and a third primer pair as shown in SEQ ID No: 7 and SEQ ID No: 8;

[0015] And / or, the prokaryotic expression vector used in the construction of the recombinant plasmid is pGEX-4T-1.

[0016] The present invention also provides an application of the above-mentioned avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein or the above-mentioned avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein expressed by the recombinant plasmid. The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is applied to the preparation of avian pathogenic Escherichia coli vaccine and / or Newcastle disease virus vaccine.

[0017] Preferably, the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is used in the preparation of a double vaccine of avian pathogenic Escherichia coli and Newcastle disease virus.

[0018] The technical solution of the present invention has at least the following advantages:

[0019] 1. The present invention successfully prepared the chimeric protein of avian pathogenic Escherichia coli OmpA-Newcastle disease virus F by using a prokaryotic expression system. The chimeric protein has similar immunogenicity to natural avian pathogenic Escherichia coli OmpA and Newcastle disease virus F protein, and can specifically bind to APEC and NDV antibodies. When the chimeric protein is used for immunization, it can produce a dual immune protection effect against APEC and NDV infection, and is suitable for the development of a new APEC and NDV double vaccine.

[0020] 2. The polyclonal antibody prepared by the present invention, obtained by immunizing animals with the chimeric protein of avian pathogenic Escherichia coli OmpA-Newcastle disease virus F as an antigen, has dual specificity for recognizing Escherichia coli OmpA and Newcastle disease virus F protein.

[0021] 3. The avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein of the present invention can be achieved based on plasmid recombination and induced expression. The preparation method is simple, suitable for industrial large-scale production, can achieve product standardization, and is conducive to comparison of results between different laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 This is a carbohydrate gel electrophoresis diagram of the recombinant plasmid identified and prepared in Example 1 of the present invention. Lane M is a molecular weight marker, lane 1 is the undigested recombinant plasmid, and lane 2 is the digested product of the recombinant plasmid;

[0024] Figure 2 The expression of the chimeric protein detected by SDS-PAGE in Example 2 of the present invention. Lane M is a molecular weight marker, lane 1 is a bacterial culture induced by pGEX-4T-1 / BL21 (Rosetta), lane 2 is a bacterial culture not induced by pGEX-4T-1 / BL21 (Rosetta), lane 3 is a bacterial culture induced by pGEX-O1-Fo-O2 / BL21 (Rosetta), lane 4 is a supernatant of ultrasonic fragmentation of a bacterial culture induced by pGEX-O1-Fo-O2 / BL21 (Rosetta), and lane 5 is a precipitate of ultrasonic fragmentation of a bacterial culture induced by pGEX-O1-Fo-O2 / BL21 (Rosetta);

[0025] Figure 3 The purification of the chimeric protein detected by SDS-PAGE in Example 2 of the present invention. Lane M is a molecular weight marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is a purified chimeric protein sample;

[0026] Figure 4 The purification of polyclonal antibodies detected by SDS-PAGE in Application Example 1 of the present invention is shown in FIG. 1. Lane M is a molecular weight marker, lane 1 is a polyclonal antibody obtained by the first round of column purification, and lane 2 is a polyclonal antibody obtained by the second round of column purification. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0028] The technical scheme of the present invention is described in detail by way of examples below. Among them, the nucleotide sequence of the first coding gene fragment (684 bp) is shown in SEQ ID No: 9, the nucleotide sequence of the second coding gene fragment (294 bp) is shown in SEQ ID No: 10, and the nucleotide sequence of the third coding gene fragment (1089 bp) is shown in SEQ ID No: 11, and is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The construction of the recombinant plasmid adopts The cloning was performed using the IIOne Step cloning kit, and the three primer pairs used were synthesized by Invitrogen (Shanghai) Trading Co., Ltd.

[0029] In the present invention, the prepared chimeric protein is specifically the avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein.

[0030] Example 1, preparation of recombinant plasmid:

[0031] The first coding gene fragment (denoted as OmpA1), the third coding gene fragment (denoted as Fo), and the second coding gene fragment (denoted as OmpA2) were sequentially connected in series and inserted into the prokaryotic expression vector pGEX-4T-1 to obtain the primary recombinant plasmid.

[0032] The primary recombinant plasmid obtained above was transformed into DH5α competent cells, and positive clones were screened on ampicillin resistance plates. The positive clone plasmid was extracted according to the instructions of AxyPrep plasmid DNA mini kit, which was the recombinant plasmid, recorded as pGEX-O1-Fo-O2. The length of the inserted gene fragment was preliminarily detected by Xho I digestion and 1% agarose gel electrophoresis. Figure 1 As shown, a DNA fragment with the expected size of 2067 bp was obtained. The clones with the correct length of the inserted fragment were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing, and the positive clones with completely correct sequences were further screened. The DH5α bacteria containing the correct recombinant plasmid were preserved.

[0033] Example 2: Induced expression, purification and testing of chimeric protein:

[0034] Take 1 μL of the recombinant plasmid pGEX-O1-Fo-O2 prepared above and transform it into 100 μL of BL21 (Rosetta) competent cells, spread it on an LB plate with an ampicillin concentration of 100 mg / L, and invert and culture it at 37°C overnight. Pick a monoclonal colony on the transformation plate and inoculate it in an LB culture solution with an ampicillin concentration of 100 mg / L, shake and culture it at 37°C and 200 r / min until the bacterial OD600 is 0.6-0.8, add IPTG to the culture to a final concentration of 0.2 mmol / L, and then shake and culture it at 15°C and 200 r / min overnight to induce the expression of the chimeric protein. At the same time, set up a synchronously induced empty vector control group GEX-4T-1 / BL21 (Rosetta) and an uninduced pGEX-O1-Fo-O2 / BL21 (Rosetta) control group. After the induction expression was completed, the expression product was detected by 12% SDS-PAGE, and the protein bands in the gel were displayed by Coomassie Brilliant Blue staining. The gel was observed and the image was collected under the gel imaging system. The results were as follows: Figure 2 As shown, the target band appeared at about 102 ku, and the chimeric protein was mainly expressed in inclusion bodies.

[0035] The BL21 (Rosetta) precipitate after the above-mentioned induced expression was ultrasonically disrupted. The conditions during the disruption process were: probe power 600W, working 6s, interval 6s, and total time 12min. The urea dissolution method was used to denature the inclusion body protein, and then the inclusion body renaturation solution was affinity purified using a low-pressure chromatography system and a GST column. The protein concentration was determined using a BCA protein quantification kit. The obtained avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein was subjected to SDS-PAGE electrophoresis to detect the protein purity. Figure 3 It was found that a high-purity avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein was obtained.

[0036] Application Example 1: Preparation and Application of Rabbit Anti-Avian Chimeric Protein Polyclonal Antibodies

[0037] A. Animal Immunization:

[0038] The chimeric protein purified in Example 2 was used to subcutaneously immunize two New Zealand white rabbits, once every two weeks, with 400 μg per time, for a total of three immunizations.

[0039] B. Preparation of polyclonal antibodies:

[0040] One day before the first immunization, blood was collected from the ear vein of the rabbit, and the blood was placed at 37°C for 1 hour, serum was separated at 4°C overnight, and red blood cells were removed by centrifugation at 5000r / min for 10 minutes, and serum was collected as a negative serum control. Ten days after the third immunization, blood was collected from the carotid artery, blood was placed at 37°C for 1 hour, serum was separated at 4°C overnight, red blood cells were removed by centrifugation at 5000r / min for 10 minutes, and serum was collected. First, amino gel was coupled with avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein to prepare an antigen affinity purification chromatography column, and the antiserum was purified by the first round of column purification, eluted with glycine elution buffer and dialyzed. Then, amino gel was coupled with GST to prepare an antigen affinity purification chromatography column, and the antiserum after the first round of column was passed through the second round of column to obtain antibodies with cross-reactive components removed, and dialyzed again to obtain polyclonal antibodies, which were recorded as O1FoO2 antibodies. The concentration of the purified polyclonal antibody was detected using a BCA protein concentration assay kit, and the antibody purity was further tested by SDS-PAGE electrophoresis. Figure 4 It can be seen that the polyclonal antibody obtained after column purification has a high purity, and clear antibody heavy chain and light chain bands can be seen, with sizes of 55ku and 25ku respectively.

[0041] C. Detection of polyclonal antibodies:

[0042] The prepared polyclonal antibody was used to determine the titer of the polyclonal antibody against the chimeric protein by the indirect ELISA method. The antigen (avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein) was coated on the ELISA plate at a coating concentration of 5 ng / μL, 100 μL per well. The antibody was diluted 3 times from 1:4500, 100 μL per well. The enzyme-labeled secondary antibody was HRP-labeled goat anti-rabbit IgG (1:50000 dilution). Analysis of the indirect ELISA test results (see Table 1) showed that the titer of the chimeric protein rabbit polyclonal antibody was approximately 1.1×10 6 .

[0043] Table 1

[0044]

[0045] The bispecificity of the prepared polyclonal antibody was determined by Western blot method. According to the test, the polyclonal antibody can specifically bind to OmpA protein in avian pathogenic Escherichia coli and F protein in Newcastle disease virus respectively.

[0046] Application Example 2: Immune protection effect of avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein on chicks

[0047] A. Vaccine preparation:

[0048] The avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein was diluted with sterile saline to a concentration of 10 mg / mL for animal inoculation.

[0049] Inoculate LB liquid culture medium with avian pathogenic Escherichia coli of serotype O18, culture at 37°C with shaking for 10 hours, add 0.4% formalin by volume after purity test and bacterial count, and shake at 37°C and 200 rpm for 24 hours for inactivation; spread 200 μL of the inactivated bacterial solution on an LB plate, culture at 37°C for 24 hours, and perform a sterility test. No bacteria should grow.

[0050] B. Experimental grouping and immunization procedures:

[0051] Forty healthy Roman hens aged 7 days were randomly divided into four groups with 10 in each group, namely: group A1 immunized with avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein and then challenged, group A2 immunized with O18 avian pathogenic Escherichia coli inactivated vaccine and then challenged, control group C1 challenged with O18 avian pathogenic Escherichia coli, and control group C2 not immunized and not challenged. The immunization route was subcutaneous injection in the neck. The specific immunization procedures of the groups are shown in Table 2.

[0052] Table 2

[0053]

[0054] C. Protection of chicks from poison attack:

[0055] 14 days after the second vaccination, the A1, A2 and C1 groups of chickens were challenged with a virulent strain of avian pathogenic Escherichia coli O18 (1×108 CFU / chicken) by intramuscular injection, and the C2 normal control group was treated with sterilized saline. The incidence and mortality within one week after the challenge were observed and recorded. One week after the challenge, the surviving chickens were killed and the pathological changes of the internal organs were recorded. If fibrinous pericarditis, perihepatitis, peritonitis, air sacitis or one of them appeared, it was recorded as an "infected chicken". The results are shown in Table 3.

[0056] Protection rate = (number of attacks - number of cases) ÷ number of attacks × 100%

[0057] Table 3

[0058]

[0059] From the results of the challenge protection test (Table 3), it can be seen that the immune protection rates of ducklings immunized with avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein and APEC inactivated vaccine against APEC attack were 70% and 90%, respectively, while 100% of non-immunized chickens became ill, that is, avian pathogenic Escherichia coli OmpA-Newcastle disease virus F chimeric protein can be used as a genetically engineered subunit vaccine to prevent APEC.

[0060] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0061] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0062] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein, characterized in that: The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is obtained by inducing expression of a gene encoding an outer membrane protein A of avian pathogenic Escherichia coli and a gene encoding a Newcastle disease virus F protein after recombination.

2. The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein according to claim 1, characterized in that: The amino acid sequence of the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is shown in SEQ ID No:

1.

3. A recombinant plasmid for expressing the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein according to claim 1 or 2, characterized in that: The recombinant plasmid comprises a vector and a target gene, and the target gene comprises a gene encoding an outer membrane protein A of avian pathogenic Escherichia coli and a gene encoding a Newcastle disease virus F protein.

4. A recombinant plasmid according to claim 3, characterized in that: The target gene is obtained by inserting the third coding gene fragment of the longest outer membrane region of Newcastle disease virus F protein between the first coding gene fragment of the N-terminal part of the outer membrane region of avian pathogenic Escherichia coli outer membrane protein A and the second coding gene fragment of the C-terminal part of the outer membrane region of avian pathogenic Escherichia coli outer membrane protein A.

5. A recombinant plasmid according to claim 4, characterized in that: The length of the first coding gene fragment is 684 bp; And / or, the length of the second coding gene fragment is 294 bp; And / or, the length of the third coding gene fragment is 1089 bp.

6. A recombinant plasmid according to claim 5, characterized in that: The nucleotide sequence of the target gene is shown in SEQ ID No:

2.

7. A recombinant plasmid according to claim 3, characterized in that: The primers used in the construction of the recombinant plasmid include a first primer pair as shown in SEQ ID No: 3 and SEQ ID No: 4, a second primer pair as shown in SEQ ID No: 5 and SEQ ID No: 6, and a third primer pair as shown in SEQ ID No: 7 and SEQ ID No: 8; And / or, the prokaryotic expression vector used in the construction of the recombinant plasmid is pGEX-4T-1.

8. A use of the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein according to claim 1 or 2 or the avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein expressed by the recombinant plasmid according to claims 3-7, characterized in that: The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is used in the preparation of avian pathogenic Escherichia coli vaccine and / or Newcastle disease virus vaccine.

9. A use according to claim 8, characterized in that: The avian pathogenic Escherichia coli-Newcastle disease virus chimeric protein is used in the preparation of a double vaccine of avian pathogenic Escherichia coli and Newcastle disease virus.

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