CN101870733B - Recombinant Fusion Protein of Avian IL-2 and Newcastle Disease Virus HN Gene and Its Application - Google Patents

Abstract

The invention relates to a preparation method and application of recombinant avian interleukin 2 and Newcastle disease virus hemagglutinin-neuraminidase (HN) fusion protein (chIL-2-HN fusion protein), which belongs to bioengineering technology field. The fusion protein is a fusion of avian IL-2 protein and Newcastle disease virus HN protein through a flexible Linker peptide. The DNA sequence encoding the fusion protein is inserted into the expression vector pPICZαA, and yeast X-33 is electrotransformed to obtain high-efficiency expression of recombinant chIL-2 -HN fusion protein genetically engineered bacteria, recombinant chIL-2-HN fusion protein obtained through liquid culture and purification, this recombinant fusion protein can be used as a new genetic engineering vaccine for Newcastle disease, and can also be used as a new immune adjuvant for Newcastle disease conventional vaccines agent. The chIL-2-HN fusion protein of the present invention is confirmed by animal immunization experiments, has good safety and no toxic or side effects. It can effectively enhance the level of cellular immunity and humoral immunity of the body, and has broad application prospects.

Description

Recombinant Fusion Protein of Avian IL-2 and Newcastle Disease Virus HN Gene and Its Application

technical field

The present invention relates to the fusion protein of poultry interleukin-2 (Interleukin-2, IL-2) and Newcastle disease virus HN, the DNA sequence encoding the fusion protein, the vector and the host cell containing the DNA sequence, and the recombinant fusion The protein and its application belong to the technical field of genetic engineering production of vaccines and immune adjuvants in the biotechnology pharmaceutical industry.

Background technique

Newcastle disease (ND) is a high-level contact disease of poultry caused by Newcastle disease virus (NDV), characterized by dyspnea, yellow-green loose stools, neurological disorders, and mucosal and serosal hemorrhage. sexual, acute septic infectious diseases. Listed by the International Office of Epizootics as one of the most harmful Class A infectious diseases to animals, it has caused huge economic losses to the poultry industry. Newcastle disease is the number one disease that threatens the poultry industry due to its high ND morbidity and mortality. Whether the disease is successfully controlled directly determines the development trend of the poultry industry.

The HN protein encoded by the NDV-HN gene plays an important role in the pathogenic process of NDV. The HN protein, the hemagglutinin-neuraminidase protein, is another larger glycoprotein of NDV besides the F glycoprotein, and is also the main host protective antigen of the virus. HN has two activities of HA and NA, one is to adsorb receptors containing sialic acid on the cell surface; the other is to catalyze the cleavage of sialic acid receptors by NA, these two activities play a role in the recognition of cell receptors during virus infection. body and mediate the important role of virus adsorption to the cell membrane. HN protein is the main viral protein that induces the body to produce neutralizing antibodies, and plays an important role in the body's anti-infection immunity. The HN protein expressed by genetic engineering has good immunogenicity, and can produce a higher level of specificity after immunizing chickens. Sexual antibodies have a good protective effect against virulent attacks.

Vaccines routinely used in Newcastle disease immunization mainly include attenuated vaccines with different virulence and inactivated oil emulsion vaccines. However, the common feature of traditional Newcastle disease inactivated vaccines is that they can induce a strong humoral immune response, while the cellular immune response is relatively weak. In the anti-infection immunity of Newcastle disease, the antibody response is necessary for the body to resist the infection of the virus, but it cannot prevent the spread of the virus. Therefore, with the rapid development of molecular biology technology in recent years, the development of NDV genetically engineered vaccines has become a hotspot of research, but there are still some unsatisfactory aspects of genetically engineered vaccines in terms of inducing the body's immune response. Animal protection experiments The effect is not ideal. Therefore, researchers are trying to enhance the immune effect of NDV vaccine in many ways, and the use of cytokines as molecular immune adjuvants has attracted the attention of researchers.

Interleukin-2 (IL-2) is a lymphokine secreted by T lymphocytes, which can promote the proliferation and differentiation of T cells and B cells, and can enhance the killing activity of monocytes and NK cells , plays an important regulatory role in the process of immune response. Studies have found that IL-2 can specifically induce the body's cellular immunity, and non-specifically act on the body's humoral immunity. When mixed with antigenic substances, it can make the body generate immunity in advance, reduce stress response, reduce immune damage, and prolong the maintenance time of effective antibodies. , is a natural immune booster.

Contents of the invention

The purpose of the present invention is to provide a chIL-2-HN fusion protein with good immune effect.

The purpose of the present invention is also to provide a DNA sequence encoding the fusion protein.

The object of the present invention is also to provide a vector and a host cell containing the DNA sequence.

The object of the present invention is also to provide a preparation method of the genetically engineered chIL-2-HN fusion protein using Pichia pastoris as a host cell.

In addition, the object of the present invention is also to provide an application of the fusion protein in the prevention and treatment of chicken Newcastle disease.

In order to achieve the above object, the technical scheme of the present invention adopts a chIL-2-HN fusion protein, including the amino acid sequence of avian interleukin 2 (chIL-2), Newcastle disease virus hemagglutinin-neuraminidase (HN protein), and the flexible Linker peptide amino acid sequence between the amino acid sequence of avian interleukin 2 and the amino acid sequence of Newcastle disease virus HN protein; the avian interleukin 2 (chIL-2) gene and chicken Newcastle disease virus HN gene It is connected in series by a flexible Linker peptide (G-G-G-G-S).

The protein has the amino acid sequence shown in SEQ ID NO.7.

The flexible Linker peptide has the amino acid sequence shown in SEQ ID NO.6.

Simultaneously, the technical solution of the present invention also adopts a kind of isolated DNA sequence, coding has the fusion protein of the aminoacid sequence shown in SEQ ID NO.7.

The DNA sequence has the nucleotide sequence shown in SEQ ID NO.5.

Technical scheme of the present invention has also adopted a kind of recombinant Pichia vector and host cell, and it contains the DNA sequence with the nucleotide sequence shown in SEQ ID NO.5.

Application of recombinant fusion protein in prevention and treatment of chicken Newcastle disease.

Specifically: the chIL-2-HN fusion protein of the present invention and its DNA sequence are as follows:

The chIL-2-HN fusion protein gene is connected in series by the poultry IL-2 gene and chicken Newcastle disease virus HN gene through a flexible Linker (G-G-G-G-S) gene, and the amino acid sequence encoded by the tandem DNA sequence is the chIL-2-HN fusion protein . On the one hand, the HN protein of Newcastle disease virus can induce the body to produce high levels of specific antibodies, which has a good protective effect against virulent attacks. At the same time, interleukin-2 can promote the proliferation and differentiation of T cells and B cells, and can specifically induce the body's cellular immunity. The two are connected by a flexible amino acid peptide, and can effectively exert their respective biological functions while maintaining an independent spatial structure.

The present invention also adopts a method for constructing a genetically engineered bacterium capable of efficiently expressing the chIL-2-HN fusion protein:

For the chIL-2-HN fusion protein with low production cost, a kind of genetically engineered bacterium capable of producing chIL-2-HN fusion protein must be constructed. The genetically engineered bacterium selected in the present invention is Pichia pastoris X-33 bacterium, and the recombinant Pichia The gene of chIL-2-HN fusion protein was integrated into the genome of Pichia pastoris X-33 through homologous recombination technology, so the recombinant Pichia pastoris X-33 strain genome carried Gene for chIL-2-HN fusion protein.

The present invention also adopts a preparation method of a soluble recombinant chIL-2-HN fusion protein:

Using the Pichia pastoris expression system, in the BMMY medium, the recombinant Pichia pastoris X-33 strain can highly express the chIL-2-HN fusion protein under the induction of methanol; the culture supernatant is collected and subjected to Ni column affinity chromatography After purification, the breakthrough peaks were collected and freeze-dried to obtain recombinant chIL-2-HN fusion protein with extremely high purity.

The invention immunizes chickens with recombinant chIL-2-HN fusion protein. Through the determination of NDV-HN antibody and antibody subtype, spleen lymphocyte proliferation, serum IL-4 and IFN-γ of immunized chickens and animal challenge protection experiment. The immunological properties of recombinant chIL-2-HN were evaluated.

The preparation method of recombinant chIL-2-HN fusion protein of the present invention, its production comprises the following steps:

(a) obtaining the gene sequence encoding the chIL-2-HN fusion protein;

① Obtain the poultry IL-2 gene sequence:

According to the nucleotide sequence of avian IL-2 in GenBank (accession number: AY029588), DNA Star and primer premier software were used to design two primer fragments F ₁ and F ₂ to amplify the avian IL-2 gene. Primers were synthesized by Dalian Bao Biological Engineering Company. A Kpn I endonuclease site was introduced at the 5' end of primer F1, and a flexible linker was introduced at the 5' end of primer F2.

The primer sequences are as follows:

F1: SEQ ID NO.1;

5'-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3'

F2: SEQ ID NO.2;

5'-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3'

The NDV-I series vaccine was diluted 50 times, inoculated into 10-day-old chicken embryos through the allantoic cavity, 0.2 mL/piece, and incubated at 37 °C. Undead chicken embryos were taken out 48-72 hours after inoculation, the spleen was aseptically ground, and the total mRNA was extracted and amplified immediately according to the requirements of the kit. Chicken IL-2 gene was amplified by RT-PCR using the total mRNA of chicken embryo spleen as template. According to the requirements of the RT-PCR kit, add 5 μL of 10 × buffer, 10 μL of MgCl2, 5 μL of dNTP mixture, 1 μL of RNase inhibitor, 1 μL of reverse transcriptase, and 1 μL of Taq enzyme into the 50 μL reaction system, upstream and downstream Primer F1 and F2 each 2 μL, template 5 μL, RNase-free water 18 μL. Reverse transcription at 50°C for 30 min, inactivation of reverse transcriptase at 94°C for 2 min. Amplification by touchdown PCR, the parameters are: 94°C for 50 s, 58°C for 45 s, 72°C for 100 s, cycle 6 times, 94°C for 50 s, 56°C for 50 s, 72°C for 100 s, cycle 27 times, 72°C Extend for 10 min. The amplified PCR product was identified by electrophoresis on 1% agarose gel containing ethidium bromide (EB), cut out the target band, and then recovered according to the instructions of the gel recovery kit (Dalian TaKaRa Company). Avian IL-2 gene fragment;

② Obtain the HN gene of Newcastle disease virus

According to the nucleotide sequence of the Newcastle disease virus HN gene in GenBank (accession number: GU573799.1), two primer fragments F ₃ and F ₄ were designed using DNA Star and primer premier software to amplify the Newcastle disease virus HN gene, and the primer F3 5 A flexible linker was introduced at the ' end, and a Not I endonuclease site was introduced at the 5' end of F4. The primer sequences are as follows:

F3: SEQ.ID.NO.3;

5'- GGCGGCGGCGGCAGCATGGACCGTGTAGTTAGC-3'

F4: SEQ.ID.NO.4;

5'-TAGCGGCCGCAACTCTATCATCCTTGAGGATCTCAAC-3'

The frozen NDV strain was thawed in a 37°C water bath, and then inoculated into 9-10 day-old non-immune chicken embryos (0.2 mL per embryo) through the allantoic cavity, and incubated at 37°C. The chicken embryos that died within 24 hours were discarded, and the allantoic fluid of chicken embryos that died within 24 to 48 hours was aseptically collected (the chicken embryo fluid with red blood cells or egg yolk that made the allantoic fluid cloudy was discarded), and after repeated freezing and thawing three times, Centrifuge at 8 000r/min at 4°C for 15 minutes, take the supernatant, and store it at 20°C until use. The extraction of viral RNA was performed according to the instructions of the RNA extraction kit from Dalian Bao Biological Engineering Co., Ltd. Take 4 μL of extracted total RNA as a template, add 3 μL of MgCl ₂ , 2.5 μL of 10 × reverse transcription buffer, 2.5 μL of dNTP Mixture (10 mmol/L), 0.5 μL of Rnase Inhititor (20U), 0.5 μL of RT enzyme, and Taq enzyme 0.5 μL, primer F3 1 μL, primer F4 1 μL, sterilized dH ₂ O 9.5 μL, total system 25 μL. Carry out RT-PCR reaction according to the following reaction conditions: 50 ℃ 40 s, 94 ℃ 2 min, 94 ℃ 60 s, 55 ℃ 60 s, 72 ℃ 2 min 11 cycles; 94 ℃ 60 s, 53 ℃ 60 s, 72 ℃ 12 cycles of 2 min; 10 cycles of 94 °C for 60 s, 51 °C for 60 s, 72 °C for 2 min; 72 °C for 10 min. After the PCR product was identified by electrophoresis on a 1% agarose gel containing ethidium bromide, the target band was excised, and then recovered according to the instructions of the gel recovery kit, which was the Newcastle disease virus HN gene;

③ Overlap PCR amplification of gene sequence of chIL-2-HN fusion protein

Using the avian IL-2 gene and Newcastle disease virus HN gene obtained in the above ① and ② as templates, use F1 and F4 primers to amplify the chIL-2-HN fusion gene by overlapping PCR.

PCR reaction system 50 μl: 10×PCR Buffer, 5 μL, MgCl ₂ , 3 μL; dNTP, 10 mmol/L, 1 μL; poultry IL-2 gene and Newcastle disease virus HN gene were added as templates, 5 μL each, primer F1 and primer F4, and finally The concentration is 20 pmol/L, 2 μL each; TaKaRa ExTaq 0.5 μL; Sterilized ultrapure water, 34.5 μL;

PCR reaction conditions: pre-denaturation at 94°C for 2 minutes, enter PCR cycle: 94°C for 30s, annealing temperature from 55°C, 1 minute per cycle, 30 cycles in total; After gel electrophoresis identification, cut out the target band, and then recover according to the instructions of the gel recovery kit, which is the chIL-2-HN fusion gene;

(b) Insert the gene sequence of the chIL-2-HN fusion protein obtained in step (a) into the Pichia pastoris vector pPICZαA to obtain a recombinantly expressed Pichia pastoris vector, named pPICZαA-chIL-2-HN;

(c) Construction of genetically engineered strains highly expressing chIL-2-HN fusion protein

The above chIL-2-HN fusion gene PCR product and Pichia pastoris expression vector pPICZαA were digested with Kpn Ⅰ and Not I, ligated with T4 DNA Ligase, the ligated product was transformed into E.coli DH5α, and the recombinant expression plasmid was subjected to Kpn Ⅰ and Not I I double enzyme digestion identification; enzyme digestion and PCR identification of positive plasmid sequencing, named pPICZαA-chIL-2-HN; using electroporation method, the recombinant plasmid pPICZαA-chIL-2-HN was transformed into Pichia pastoris X- In 33, through high resistance screening and PCR identification, the genetically engineered Pichia pastoris X-33 strain expressing the recombinant fusion protein was obtained;

(d) Obtaining soluble chIL-2-HN fusion protein

Inoculate the above-mentioned recombinant Pichia pastoris X-33 bacteria carrying the chIL-2-HN fusion protein gene into 5 mL BMGY, shake and culture at 30°C 230r/min for about 22 hours until the OD ₆₀₀ reaches 3~6; room temperature 3000r Centrifuge at 250r/min for 2 min, collect the bacteria and resuspend them in 25 mL BMMY medium, and induce expression at 28°C, 250r/min for 60h, during which methanol with a final concentration of 1% was added every 24h; after 72h, 5000r/min Centrifuge for 10 min to collect the culture supernatant, which is the obtained chIL-2-HN fusion protein.

The recombinant chIL-2-HN fusion protein of the present invention can effectively enhance the humoral and cellular immune responses of the body. The present invention uses the recombinant chIL-2-HN fusion protein to immunize chickens. The results of antibody subtype determination show that chIL-2-HN fusion Chickens in the protein immunized group could produce a balanced IgG1 and IgG2a antibody response. The results of cytokine ELISA test showed that the fusion protein immune group mainly stimulated Th1 differentiation (FNI-g), but also stimulated Th2 (IL-4) differentiation.

Newcastle disease virus challenge test showed that chIL-2-HN fusion protein can significantly improve the protection rate of the body against virus, indicating that the recombinant chIL-2-HN fusion protein can effectively enhance the body's humoral and cellular immune responses.

The present invention is easy to realize mass production: the recombinant chIL-2-HN fusion protein of the present invention is produced by selecting the Pichia pastoris expression system, which has the advantages of simple technology, low cost, high yield, large-scale fermentation, and easy mass production.

The chIL-2-HN fusion protein designed and developed by the present invention not only has the ability to produce higher levels of specific antibodies after immunizing chickens, but also has a good protective effect against virulent attacks; in addition, it also has cytokines of interleukin-2 Immunological adjuvant effect. The present invention provides a feasible technical route for preparing recombinant chIL-2-HN fusion protein, and the chIL-2-HN fusion protein prepared according to the method can be used as a new type of genetic engineering vaccine for Newcastle disease, and can also be used as a new type of Newcastle disease conventional vaccine. The immune adjuvant has broad application prospects in the prevention and treatment of Newcastle disease.

Description of drawings

Fig. 1 Enzyme digestion and PCR identification of recombinant plasmid pPICZαA-chiIL2-HN;

Lane 1-2. Negative control; Lane3. PCR amplification product; 4. Kpn I/ Not I double digestion product; Lane M. λDNA/ EcoR I+ Hind III marker

Fig. 2 PCR identification of transformant genomic DNA;

Lane 1. Negative control; Lane 2.X-33 PCR amplification product; Lane 3.X-33/pPICZαA PCR amplification product; Lane 4.X-33/pPICZαA-chiIL2-HN PCR amplification product

Figure 3 SDS-PAGE analysis of the expression product in the recombinant yeast fermentation supernatant;

Lane 1.X-33/pPICZαA; Lane 2-6.X-33/pPICZαA-chiIL2-HN Fermentation supernatant at 0, 24, 48, 72, 96 h; Lane M. Low molecular weight protein Marke

Western blot analysis of expression product in Fig. 4 recombinant yeast fermentation supernatant;

Lane 1. X-33/pPICZαA; Lane 2-6 X-33pPICZαA-IL2-IL2-HN induced supernatant at 0, 24, 48, 72, 96 h; Lane M. Pre-stained low molecular weight protein marker

Figure 5a, Figure 5b, and Figure 5c are NDV-HN antibody and antibody subtype analysis;

Figure 6 The proliferation of lymphocytes after immunization with recombinant proteins;

Figure 7 Cytokine detection.

Detailed ways:

Example 1

Obtain the gene sequence encoding chIL-2-HN fusion protein:

According to the nucleotide sequence of avian IL-2 in GenBank (accession number: AY029588), DNA Star and primer premier software were used to design two primer fragments F ₁ and F ₂ to amplify the avian IL-2 gene. Primers were synthesized by Dalian Bao Biological Engineering Company. A Kpn I endonuclease site was introduced at the 5' end of primer F1, and a flexible linker was introduced at the 5' end of primer F2. The primer sequences are as follows:

F1: SEQ ID NO.1;

5'-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3'

F2: SEQ ID NO.2;

5'-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3'

The NDV-I series vaccine was diluted 50 times, inoculated into 10-day-old chicken embryos through the allantoic cavity, 0.2 mL/piece, and incubated at 37 °C. Undead chicken embryos were taken out 48-72 hours after inoculation, the spleen was aseptically ground, and the total mRNA was extracted and amplified immediately according to the requirements of the kit. Chicken IL-2 gene was amplified by RT-PCR using the total mRNA of chicken embryo spleen as template. According to the requirements of the RT-PCR kit, add 5 μL of 10 × buffer, 10 μL of MgCl2, 5 μL of dNTP mixture, 1 μL of RNase inhibitor, 1 μL of reverse transcriptase, and 1 μL of Taq enzyme into the 50 μL reaction system, upstream and downstream Primer F1 and F2 each 2 μL, template 5 μL, RNase-free water 18 μL. Reverse transcription at 50°C for 30 min, inactivation of reverse transcriptase at 94°C for 2 min. Amplification by touchdown PCR, the parameters are: 94°C for 50 s, 58°C for 45 s, 72°C for 100 s, cycle 6 times, 94°C for 50 s, 56°C for 50 s, 72°C for 100 s, cycle 27 times, 72°C Extend for 10 min. The amplified PCR product was identified by electrophoresis on 1% agarose gel containing ethidium bromide (EB), cut out the target band, and then recovered according to the instructions of the gel recovery kit (Dalian TaKaRa Company). Avian IL-2 gene fragment.

According to the nucleotide sequence of the Newcastle disease virus HN gene in GenBank (accession number: GU573799.1), two primer fragments F ₃ and F ₄ were designed using DNA Star and primer premier software to amplify the Newcastle disease virus HN gene, and the primer F3 5 A flexible linker was introduced at the ' end, and a Not I endonuclease site was introduced at the 5' end of F4. The primer sequences are as follows:

F3: SEQ.ID.NO.3;

5'- GGCGGCGGCGGCAGCATGGACCGTGTAGTTAGC-3'

F4: SEQ.ID.NO.4;

5'-TAGCGGCCGCAACTCTATCATCCTTGAGGATCTCAAC-3'

The frozen NDV strain was thawed in a 37°C water bath, and then inoculated into 9-10 day-old non-immune chicken embryos (0.2 mL per embryo) through the allantoic cavity, and incubated at 37°C. The chicken embryos that died within 24 hours were discarded, and the allantoic fluid of chicken embryos that died within 24 to 48 hours was aseptically collected (the chicken embryo fluid with red blood cells or egg yolk that made the allantoic fluid cloudy was discarded), and after repeated freezing and thawing three times, Centrifuge at 8 000r/min at 4°C for 15 minutes, take the supernatant, and store it at 20°C until use. The extraction of viral RNA was performed according to the instructions of the RNA extraction kit from Dalian Bao Biological Engineering Co., Ltd. Take 4 μL of extracted total RNA as a template, add 3 μL of MgCl ₂ , 2.5 μL of 10 × reverse transcription buffer, 2.5 μL of dNTP Mixture (10 mmol/L), 0.5 μL of Rnase Inhititor (20U), 0.5 μL of RT enzyme, and Taq enzyme 0.5 μL, primer F3 1 μL, primer F4 1 μL, sterilized dH ₂ O 9.5 μL, total system 25 μL. Carry out RT-PCR reaction according to the following reaction conditions: 50 ℃ 40 s, 94 ℃ 2 min, 94 ℃ 60 s, 55 ℃ 60 s, 72 ℃ 2 min 11 cycles; 94 ℃ 60 s, 53 ℃ 60 s, 72 ℃ 12 cycles of 2 min; 10 cycles of 94 °C for 60 s, 51 °C for 60 s, 72 °C for 2 min; 72 °C for 10 min. After the PCR product was identified by electrophoresis on 1% agarose gel containing ethidium bromide, the target band was excised, and then recovered according to the instructions of the gel recovery kit, which was the Newcastle disease virus HN gene.

Using the avian IL-2 gene and NDV HN gene obtained above as templates, the chIL-2-HN fusion gene was amplified by overlapping PCR using primers F1 and F4.

PCR reaction system 50 μl: 10×PCR Buffer, 5 μL, MgCl ₂ , 3 μL; dNTP, 10 mmol/L, 1 μL; avian IL-2 gene and Newcastle disease virus HN gene were added as templates in 5 μL each, primer F1 and primer F4 were added, and finally The concentration is 20 pmol/L, 2 μL each; TaKaRa ExTaq 0.5 μL; Sterilized ultrapure water, 34.5 μL;

PCR reaction conditions: pre-denaturation at 94°C for 2 minutes, enter PCR cycle: 94°C for 30s, annealing temperature from 55°C, 1 minute per cycle, 30 cycles in total; After identification by gel electrophoresis, cut out the band of interest, and then recover according to the instructions of the gel extraction kit, which is the chIL-2-HN fusion gene. (SEQ. ID. NO. 5).

Example 2

Construction of chIL-2-HN Fusion Gene Recombinant Yeast Expression Vector

Use Kpn I and Not I endonucleases to perform double enzyme digestion on the PCR product of the above chIL-2-HN fusion gene and Pichia pastoris expression vector pPICZαA, and place them in a water bath at 37 °C for 2 h. After the sugar gel electrophoresis identification, the gel recovery kit is used for recovery identification. The digested chIL-2-HN fusion gene and Pichia pastoris expression vector pPICZαA were connected overnight at 4°C at a molar ratio of 1:3. The ligated product was added to a polypropylene centrifuge tube containing 100 μL of competent DH5α, mixed gently, and then placed in an ice bath for 30 min. After the polypropylene centrifuge tube was taken out of the ice, it was heat-shocked at 42 °C for 90 sec, and then immediately placed in an ice bath for 2 min. Add 800 μL of LB medium preheated at 37 °C, and shake (100-150 rpm) at 37 °C for 45 min. Take 100 μL of the bacterial solution and spread evenly on the agar LB plate containing 50 μg/mL of ampicillin (Amp), place it upright at 37 °C for 20 min, and then incubate it upside down for 16-20 h. Extract the plasmid according to the alkaline lysis method on the "Molecular Cloning Experiment Guide". The extracted plasmid was identified by Kpn I and Not I double enzyme digestion. The plasmid with a DNA fragment of about 2000 bp in size after enzyme digestion was regarded as a positive plasmid (Figure 1). And the positive plasmid was named pPICZαA-chIL-2-HN. Sent to Shanghai Invitrongen Company for sequencing.

Example 3

Construction of genetically engineered strains expressing chIL-2-HN fusion protein:

The positive recombinant yeast expression vector pPICZαA-chIL-2-HN obtained in Example 2 was linearized with SacI, and 5 μg of the linearized recombinant expression vector pPICZαA-chIL-2-HN was mixed with 80 μL of competent Pichia pastoris X- Mix 33 phases, transfer to a pre-cooled 0.2 cm electric cup, place on ice for 5 min, 1.5 kV, 25 μF, 200 Ω electric shock, immediately add 1 mL of pre-cooled 1 mol/L sorbitol, take 200 μL and spread on On the YPDS plate, cultivate at 30°C until a single colony appears; refer to the Pichia Expression Kit for detailed steps; analyze the Pichia transformants by PCR method, prepare the PCR template by the boil-freeze-boil method, and use primers F1 and F4: reaction system Same as above, PCR reaction conditions: 94°C for 5 min; 94°C for 45 s, 48°C for 45 s, 72°C for 45 s, 25 cycles; 72°C for 6 min; identify and amplify a clone with a size of about 2200 bp as Pichia Yeast-positive transformants (Figure 2) are genetically engineered Pichia pastoris X-33 strains expressing chIL-2-HN fusion protein.

Example 4

Induced expression of recombinant chIL-2-HN fusion protein:

Inoculate the recombinant Pichia pastoris X-33 strain carrying the chIL-2-HN fusion protein gene into 5mL BMGY, culture at 230r/min at 30℃ for about 22 hours until the OD ₆₀₀ reaches 3~6; room temperature 3000r/min Centrifuge for 2 min, collect the bacteria and resuspend in 25 mL BMMY medium, and induce expression at 28°C, 250 r/min for 60 h, during which methanol with a final concentration of 1% was added every 24 h; after 72 h, 5000 r/min The supernatant of the culture medium was collected by centrifugation for 10 min, which was the obtained chIL-2-HN fusion protein. The molecular weight of chIL-2-HN fusion protein can be identified by SDS-PAGE electrophoresis gel, the specific steps are as follows:

Add 100 μL of the collected supernatant of the above culture solution to an equal volume of 2×SDS gel loading buffer (100 mmol/L Tris Cl (pH6.8); 200 mmol/L dithiothreitol (DTT) ; 4% SDS (electrophoresis grade); 0.2 % bromophenol blue; 20% glycerol), boiled at 100 ℃ for 5 min to deform the protein, took 10 μL of the sample for SDS-PAGE gel electrophoresis, SDS-PAGE electrophoresis gel Preparation and electrophoresis conditions refer to the molecular cloning manual. After the electrophoresis gel is prepared, pour Tris-glycine electrophoresis buffer (25 mmol/L Tris; 250 mmol/L glycine (electrophoresis grade) (pH8.3); 0.1% SDS) into the electrophoresis tank. Turn on the power. The negative end of the power supply is connected to the slot, and the positive end is connected to the slot. The voltage in the stacking gel was 80 V, and the voltage in the separating gel was adjusted to 120 V. Turn off the power until the sample reaches the bottom of the separating gel, take out the gel, stain it with Coomassie Brilliant Blue for 1 h, then decolorize it on a decolorizing shaker for 1-2 h, and observe the results. The recombinant chIL-2-HN fusion protein has a molecular weight of about 100 KDa (Figure 3). At the same time, Western-blot test was carried out with NDV positive serum as the primary antibody. The culture supernatant of recombinant Pichia pastoris X-33 bacteria had a specific target band in the swimming lane, while the negative control Pichia pastoris X-33 There were no bands in the loading lane of the culture supernatant of the bacteria (Figure 4), and the results showed that the recombinant chIL-2-HN fusion protein was successfully expressed and had good immunogenicity.

Example 5

Application of recombinant chIL-2-HN fusion protein:

1) Animal immunity

Chickens were immunized with the above-mentioned recombinant chIL-2-HN fusion protein, and its immune characteristics were evaluated. 200 healthy chicks aged 20 days were randomly divided into 5 groups, 40 per group. The first group was a negative control, immunized with 200 μL PBS; the second group, the third group, and the fourth group were immunized with 200 ppm rIL-2, rHN, rIL-2-HN recombinant protein (diluted with 200 μL PBS ). Immunization was carried out by chest intramuscular injection. The fifth group was the Newcastle disease I vaccine control group, which was immunized with 200 μL by nasal drops and eye drops; all groups were immunized once every two weeks, and immunized three times in total. On the 7th day, 14th day, 21st day, 28th day and 35th day after immunization, 5 hearts were randomly selected for blood collection, and the serum was separated. At the same time, the thymus of each test chicken was aseptically collected for the lymphocyte proliferation test. At the same time, one week after the last immunization, 15 test chickens in each group were challenged with F ₄₈ E ₉ standard virulence (1000 ELD ₅₀ per bird). After the challenge, they were continuously observed for 7 days, and the incidence and death of test chickens in each group were recorded. Condition.

2) Newcastle disease virus HN antibody and antibody subtype detection

The production of NDV-HN IgG antibody after immunization in each group was detected by ELISA, and it was found that the IgG antibody response could be detected in the immunized group 1 week after the first immunization. Antibody production continued to increase after the second booster. Relative to the IgG level, the rHN immunization group was slightly lower than the NDV Vaccine immunization group at the first immunization and the first booster immunization, but after the second booster immunization, the difference was not significant (p>0.05). Relative to IgG1 level and IgG2a antibody response, IgG1 antibody was the predominant antibody subtype in chickens immunized with rHN, while IgG2a antibody was the predominant antibody subtype in chickens immunized with NDV Vaccine, however, using chIL-2-HN fusion protein Chickens in the immunized group could produce a balanced IgG1 and IgG2a antibody response. Especially after the second booster, predominantly IgG1 and IgG2a antibody levels were observed, suggesting that IL-2 can contribute to the regulation of Th1-type immune responses (Fig. 5a, Fig. 5b, Fig. 5c).

3) Tetramethylazolazolium blue method (MTT) analysis

From the 1st, 3rd, and 5th weeks after the first immunization, the thymus of each experimental chicken was aseptically collected every week to detect the proliferation of thymic T lymphocytes. The results showed that the chIL-2-HN fusion protein immunization group had the strongest ability to induce the proliferation of thymic T lymphocytes at week 5, and the other groups were rIL-2 immunization group, NDV Vaccine immunization group, rHN immunization group and PBS control group. The ability of the rIL-2 immunization group to induce thymus T lymphocyte proliferation was lower than that of the NDV Vaccine immunization group at the first week and the third week, but was significantly higher than that of the NDV Vaccine immunization group at the fifth week ( p <0.05) (Fig. 6).

4) Determination of IL-4 and IFN-γ in serum

Quantitative analysis of IL-4 and IFN-γ in serum by quantitative ELISA found that the secretion of IL-4 and IFN-γ induced by chIL-2-HN fusion protein was the strongest in each immune group, and the difference was extremely significant ( p < 0.01). Second, rIL-2 immunization group, NDV Vaccine immunization group and rHN immunization group mainly induced the secretion of IL-4. It induced the secretion levels of the two cytokines to be significantly lower than that of the rIL-2 immunized group, and the difference was significant ( p <0.05) (Fig. 7).

5) The results of the virus attack test

On the 35th day after immunization, 15 remaining chickens in each immunization group were challenged with F48E9 virulence. The results are shown in Table 1. During the 7-day observation period, 5 of the 15 experimental chickens in the rHN immunized group developed the disease, and the protection rate was only 66.7%. 93.3%. Only 2 of the 15 experimental chickens in the chIL-2-HN fusion protein immunized group became ill during the 7-day observation period, and the protection rate reached 86.7% (Table 1).

Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention, although the present invention has been described in detail with reference to the above embodiments. Those skilled in the art should understand that the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the claims of the present invention.

sequence listing

<110> Henan University of Science and Technology

<120> Recombinant Fusion Protein of Avian IL-2 and Newcastle Disease Virus HN Gene and Its Application

<170> patentin version 3.3

<210> 1

<211> 32

<212> DNA

<213> Synthetic

<223> Primer F1

<400> 1

cgggtaccat gtgcaaagta ctgatctttg gc 32

<210> 2

<211> 33

<212> DNA

<213> Synthetic

<223> Primer F2

<400> 2

gctgccgccg ccgccttttt gcagatatct cac 33

<210> 3

<211> 33

<212> DNA

<213> Synthetic

<223> Primer F3

<400> 3

ggcggcggcg gcagcatgga ccgtgtagtt agc 33

<210> 4

<211> 37

<212> DNA

<213> Synthetic

<223> Primer F4

<400> 4

tagcggccgc aactctatca tccttgagga tctcaac 37

<210> 5

<211> 2154

<212> DNA

<221> Gene for chIL-2-HN fusion protein

<400> 5

atgtgcaaag tactgatctt tggctgtatt tcggtagcaa tgctaatgac tacagcttat 60

ggagcatctc tatcatcagc aaaaaggaaa cctcttcaaa cattaataaa ggatttagaa 120

atattggaaa atatcaagaa caagattcat ctcgagctct acacaccaac tgagacccag 180

gagtgcaccc agcaaactct gcagtgttac ctgggagaag tggttactct gaagaaagaa 240

actgaagatg acactgaaat taaagaagaa tttgtaactg ctattcaaaa tatcgaaaag 300

aacctcaaga gtcttacggg tctaaatcac accggaagtg aatgcaagat ctgtgaagct 360

aacaacaaga aaaaatttcc tgattttctc catgaactga ccaactttgt gagatatctg 420

caaaaaggcg gcggcggcag catggaccgt gtagttagca gagtcgtgct ggagaatgag 480

gaaagagaag caaagaacac atggcgcctg gttttccgga tcgcagtctt acttttaatg 540

gtaatgactc tagctatctc cgcagctgcc ctggcatata gcacgggggc cagtacgccg 600

catgacctcg caggcatatc gactgtgatc tccaagacag aagataaggt tacgtcttta 660

ctcagttcaa gtcaagatgt gatagatagg atatacaagc aggtggctct tgaatccccg 720

ctggcgctac taaacactga atctataatt atgaatgcaa taacctctct ttcttatcaa 780

attaacgggg ctgagaacaa tagcggatgt ggtgcgcctg ttcatgaccc agattatatc 840

ggggggatag gcaaagaact catagtggac gacaccagtg atgtcacatc attttatcct 900

tctgcatatc aagaacactt gaatttcatc ccggcgccca ccacaggatc cggttgcact 960

cggataccct catttgacat gagcgccacc cattattgtt atactcacaa tgtgatacta 1020

tccggttgca gagatcactc acactcacat caatacttag cacttggtgt gcttcggaca 1080

tctgcaacag ggagggtatt cttttctact ctgcgctcca tcaatttaga tgacacccaa 1140

aatcggaagt cctgcagtgt gagtgcaacc cctttaggtt gtgatatgct gtgctctaag 1200

gtcacaggga ctgaagagga ggattacaag tcaattgccc ccacatcaat ggtgcacgga 1260

aggctaggt ttgacggtca ataccatgag aaggacttag acaccacggt cttatttaaa 1320

gattgggtgg caaattaccc gggagtggga ggagggtctt ttattgacga ccgtgtatgg 1380

ttcccagttt acggagggct caaacccaat tcacccagtg acactgcaca agaagggaaa 1440

tatgtaatat acaagcgcca taacaacacc tgccccgatg aacaagatta ccaaattcgg 1500

atggctaagt cttcatataa acccgggcga tttggtggaa agcgcgtaca gcaagccatc 1560

ctatccatca aagtgtcaac atccctgggt aaggacccgg tgctgactat tccacctaat 1620

acaatcacac tcatgggagc cgaaggcaga atcctcacag tagggacatc tcacttcttg 1680

taccaacgag ggtcttccta tttctcccct gccttattat atcccatgac agtaaataac 1740

aaaacggcta cactccatag tccttacacg tttaatgctt tcactcggcc aggtagtgtc 1800

ccttgccagg catcagcaag atgccccaac tcatgcatca ctggggtcta taccgatcca 1860

tatcccttaa tcttccatag gaatcatact ctacgagggg tcttcgggac gatgcttgat 1920

gatgagcaag cgaggcttaa ccccgtatct gcggtattcg acaacatatc tcgcagtcgt 1980

gtcactcggg tgagttcaag cagcaccaag gcagcataca cgacatcgac atgttttaaa 2040

gttgtcaaga ccaataaagc ttaattgtctt agtatcgcag aaatatccaa taccctattc 2100

ggggaattca ggatcgttcc cttattagtt gagatcctca aggatgatag agtt 2154

<210> 6

<211> 5

<212> PRT

<221> Flexible Linker peptide

<400> 6

ggggs 5

<210> 7

<211> 718

<212> PRT

<221> chIL-2-HN fusion protein

<400> 7

mckvlifgci svamlmttay gaslssakrk plqtlikdle ileniknkih lelytptetq 60

ectqqtlqcy lgevvtlkke teddteikee fvtaiqniek nlksltglnh tgsecicea 120

nnkkkfpdfl heltnfvryl qkggggsmdr vvsrvvlene ereakntwrl vfriavlllm 180

vmtlaisaaa laystgastp hdlagistvi sktedkvtsl lsssqdvidr iykqvalesp 240

lallntesii mnaitslsyq ingaennsgc gapvhdpdyi ggigkelivd dtsdvtsfyp 300

sayqehlnfi papttgsgct ripsfdmsat hycythnvil sgcrdhshsh qylalgvlrt 360

satgrvffst lrsinlddtq nrkscsvsat plgcdmlcsk vtgteeedyk siaptsmvhg 420

rlgfdgqyhe kdldttvlfk dwvanypgvg ggsfiddrvw fpvygglkpn spsdtaqegk 480

yviykrhnnt cpdeqdyqir makssykpgr fggkrvqqai lsikvstslg kdpvltippn 540

titlmgaegr iltvgtshfl yqrgssyfsp allypmtvnn ktatlhspyt fnaftrpgsv 600

pcqasarcpn scitgvytdp yplifhrnht lrgvfgtmld deqarlnpvs avfdnisrsr 660

vtrvsssstk aayttstcfk vvktnkaycl siaeisntlf gefrivpllv eilkddrv 718

Claims (2)

1.chIL-2-HN fusion rotein is characterized in that: consist of: fowl leukocyte be situated between element 2 aminoacid sequence, Avian pneumo-encephalitis virus hemagglutinin-neuraminidase aminoacid sequence and in be situated between flexible Linker peptide ammino acid sequence between the aminoacid sequence of plain 2 aminoacid sequences and newcastle disease virus HN albumen of fowl leukocyte; Wherein, described flexible Linker peptide is the aminoacid sequence shown in the SEQ ID NO.6; Described chIL-2-HN fusion rotein is prepared as follows and obtains: the gene of the aminoacid sequence of coding fowl leukocyte Jie element 2 is the nucleotide sequence number of including according to fowl IL-2 among the GenBank: AY029588, utilize DNA Star, 2 primer fragments of primer premier software design F1, F2, fowl IL-2 gene is introduced by 5 ' end KpnPrimers F 2 amplifications that the primers F 1 of I restriction enzyme site and 5 ' end are introduced flexible joint obtain; Primer sequence is as follows:

F1: SEQ ID NO.1；

5’-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3’，

F2: SEQ ID NO.2；

5’-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3’；

The gene of the aminoacid sequence of coding Avian pneumo-encephalitis virus hemagglutinin-neuraminidase is the nucleotide sequence number of including according to NDV HN chimeric gene among the GenBank: GU573799.1, utilize DNA Star, 2 primer fragments of primer premier software design F3, F4, primers F 3 and 5 ' end that NDV HN chimeric gene is introduced flexible joint by 5 ' end are introduced NotPrimers F 4 amplifications of I restriction enzyme site obtain, and primer sequence is as follows:

F3: SEQ.ID.NO.3；

5’- GGCGGCGGCGGCAGCATGGACCGTGTAGTTAGC-3’，

F4: SEQ.ID.NO.4；

5’-TAGCGGCCGCaactctatcatccttgaggatctcaac-3’；

Take the fowl IL-2 gene of above-mentioned acquisition and NDV HN chimeric gene as template, utilize F1 and F4 primer, by overlapping PCR method amplification chIL-2-HN fusion gene; The chIL-2-HN fusion gene that the PCR method amplification is obtained is inserted among the pichia vector pPICZ α A, obtains recombinant expressed pichia vector, called after pPICZ α A-chIL-2-HN; With the recombinant expressed pichia vector pPICZ α A-chIL-2-HN that obtains, electric shock is transformed in the Pichia yeast X-33 competent cell, high resistance screening and PCR identify, acquisition is expressed the gene pichia pastoris X-33 bacterial strain of recombination fusion protein and is cultivated under suitable culture condition, separation and Culture liquid supernatant obtains recombination fusion protein.

2. the application of the described chIL-2-HN fusion rotein of claim 1 aspect preparation prevention and treatment newcastle disease medicine.

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