CN105671051A - Application of duck BCL2L15 gene in livestock and poultry for resisting avian influenza virus (AIV) - Google Patents

Abstract

The invention relates to the field of antiviral genes, and specifically discloses the application of duck BCL2L15 gene in livestock and poultry anti-AIV virus. The present invention provides a new gene BCL2L15 related to anti-bird flu, and for the first time uses the method of molecular biology combined with cell biology experiments to prove that BCL2L15 has the function of inhibiting AIV virus replication. The analysis showed that, compared with cells without overexpressing the BCL2L15 gene, the cells overexpressing the BCL2L15 gene can significantly inhibit the replication of the AIV virus, so in-depth functional research on the duck BCL2L15 gene can be carried out to determine its key protein domain against the AIV virus or amino acids. Use gene editing methods such as transgenic technology to obtain transgenic livestock and poultry that can inducibly express the BCL2L15 gene of ducks or other animals, and cultivate excellent varieties of transgenic agricultural animals that are resistant to various types of viruses such as AIV.

Description

Application of duck BCL2L15 gene in anti-AIV virus of livestock and poultry

technical field

The invention relates to the field of antiviral genes, in particular to the application of duck BCL2L15 gene in livestock and poultry anti-AIV virus.

Background technique

Influenza viruses are negative-strand RNA viruses containing eight RNA genome segments. Highly pathogenic avian influenza (Highly Pathogenic Avian Influenza, HPAI) is a severe infectious disease mainly in poultry caused by influenza A virus of the Orthomyxoviridae family. Since avian influenza was reported in Italy in 1878, two subtypes of H5 and H7 have continued to cause influenza outbreaks in chickens and turkeys around the world, seriously threatening the development of the poultry industry. Recent studies have shown that three of the four worldwide influenza pandemics in the last century, namely the Spanish flu in 1918, the Asian influenza A2 (H2N2) subtype in 1957, and the Hong Kong influenza A3 (H3N2) subtype in 1968, Closely related to avian influenza virus. These three worldwide influenza pandemics have brought serious disasters to mankind, social unrest and huge economic losses. The World Organization for Animal Health (OIE) lists avian influenza (AIV) as an animal infectious disease that must be reported, and my country lists it as a first-class animal disease.

Waterfowl, including domestic ducks, are the natural hosts of influenza virus, and all subtypes of different combinations of 16 HA and 9 NA could be isolated in waterfowl. Influenza virus has always maintained the characteristics of low pathogenicity to waterfowl. Waterfowl infected with the virus will not get sick, but they can shed the virus to the outside world. Certain strains with low pathogenicity to waterfowl are highly pathogenic to chickens or other hosts. However, with the continuous evolution of influenza viruses, the balance between waterfowl and influenza viruses has been broken. For example, in 2002, the H5N1 subtype strain that killed waterfowl was first reported in Hong Kong, and in 2005, a large-scale outbreak of H5N1 subtype influenza virus that killed migratory birds occurred in Qinghai Lake. There are many ways for the emergence of new strains circulating in the population: the gene rearrangement of avian influenza virus or other influenza viruses and human influenza viruses produces the epidemic strains infecting humans, the epidemic strains produced by the adaptation of avian influenza viruses in pigs, The spread of avian influenza virus to humans produces popular strains and the re-circulation of old human influenza virus strains after a lapse of several years. In addition, there is antigenic drift of human influenza virus itself.

In short, the most important feature of influenza A virus is that it has a wide range of hosts, numerous subtypes, various mutation forms, sudden onset, strong epidemic and pathogenicity, and it is easy to induce serious complications and cause great harm. Therefore, while studying the variation, evolution, prevalence and distribution of influenza viruses, and improving the ability to prevent and control influenza, scientists are also working to identify new immune genes against influenza viruses, and analyze the host's influence on the infectivity and pathogenicity of influenza viruses And the research on the molecular mechanism of immune response, in order to improve the host's disease resistance and promote the development of new means of preventing and controlling influenza virus.

Contents of the invention

In order to solve the problems in the prior art, the purpose of the present invention is to provide the application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry.

In order to realize the object of the invention, the technical scheme of the present invention is as follows:

The present invention provides the application of the BCL2L15 gene in influencing virus replication, the BCL2L15 gene is the nucleotide sequence shown in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID No.1 after substitution, deletion and/or addition of one or several A nucleotide sequence derived from SEQIDNo.1 with equivalent functions obtained from nucleotides. The amino acid sequence of the expressed protein is shown in SEQ ID NO.2.

Specifically, the application includes inhibiting virus replication in cells by overexpressing the BCL2L15 gene in cells, and a significant increase in virus replication in cells knocked out of BCL2L15.

The virus can be AIV, NDV, IBDV, etc.

Preferably, the virus is an avian influenza virus. The overexpression of BCL2L15 gene in cells infected with avian influenza virus can significantly inhibit the replication of avian influenza virus.

Further, the present invention also provides the application of BCL2L15 gene in anti-AIV virus. It should be noted that the present invention claims protection of the application of the gene in anti-AIV virus, because anti-AIV virus is not equivalent to treating bird flu, and inhibiting the replication of AIV virus does not mean that animals infected with AIV virus can be cured Therefore, the technical solution claimed in the present invention does not involve the diagnosis and treatment methods of diseases.

More specifically, the present invention provides a specific method, constructing the CDS sequence of the BCL2L15 gene into a vector capable of highly expressing foreign genes, obtaining a recombinant vector, and introducing the recombinant vector into avian DF1 cells.

Preferably, the vector is a eukaryotic expression vector containing a strong promoter, so as to realize the overexpression of the BCL2L15 gene.

More preferably, in order to better realize the overexpression of the BCL2L15 gene, its nucleotide sequence is shown in SEQ ID NO.3. The vector promotes the high expression of the target gene in eukaryotic cells through the CAG promoter, and has independent GFP and Neo double selection markers, and uses the transposon system to efficiently integrate into the cell genome.

When the above-mentioned vector is used to construct the recombinant vector, the CDS sequence of the BCL2L15 gene is inserted into the 2828bp of the sequence shown in SEQ ID NO.3.

The invention also provides a transgenic cell containing BCL2L15 gene.

Preferably, the transgenic cell is chicken embryo fibroblast immortalized cell line DF1.

The present invention also provides a tool cell, the tool cell is a cell overexpressing the BCL2L15 gene. The cells are not capable of totipotent differentiation.

The present invention also provides a tool cell for vaccine production, wherein the tool cell is a cell in which the BCL2L15 gene has been knocked out. The cells are not capable of totipotent differentiation.

The present invention also provides a method for constructing a transgenic animal, the method comprising transferring the duck BCL2L15 gene of the present invention into the animal body.

The beneficial effects of the present invention are:

The prior art only discloses that the BCL2L15 gene is involved in the process of apoptosis and is associated with cell transformation and the occurrence of gastrointestinal tumors. However, the present invention provides a theoretical basis for using the BCL2L15 gene to prepare transgenic animals resistant to viruses such as AIV.

The present invention obtains BCL2L15 gene mRNA sequencing fragments according to transcriptome data analysis results, respectively in two H5N1 strains (A/duck/Hubei/49/05, DK/49, highly pathogenic) and (A/goose/Hubei/65 /05, GS/65, low pathogenicity), the expression in the spleen, lung and brain tissue of the 4-week-old Shaoxing duck was significantly increased on the 1st, 2nd and 3rd day after infection (see Figure 1), confirming that duck BCL2L15 gene is an important candidate gene for resistance to influenza virus.

The present invention provides a new gene BCL2L15 related to anti-bird flu, and for the first time uses the method of molecular biology combined with cell biology experiments to prove that BCL2L15 has the function of inhibiting AIV virus replication.

The BCL2L15 gene of the present invention can be used to prepare transgenic animals resistant to influenza virus. In addition, the cell line knocked out of the BCL2L15 gene can be used as a tool cell for the production of virus vaccines such as AIV.

The BCL2L15 gene provided by the present invention can be used to inhibit influenza virus, especially can significantly inhibit the replication of AIV virus, so in-depth functional research can be carried out on the duck BCL2L15 gene, so as to determine its key protein domain or amino acid against AIV virus. Use gene editing methods such as transgenic technology to obtain transgenic livestock and poultry that can inducibly express the BCL2L15 gene, and cultivate excellent varieties of transgenic agricultural animals that are resistant to various types of viruses such as AIV.

Description of drawings

Fig. 1 is the duck BCL2L15 gene mRNA sequence that the present invention utilizes transcriptome data analysis to obtain, in two strains of H5N1 strains (A/duck/Hubei/49/05, DK/49, highly pathogenic) and (A/goose/ Hubei/65/05, GS/65, low pathogenicity), the expression patterns in spleen, lung and brain tissues on the 1st, 2nd and 3rd day after infecting 4-week-old Shaoxing ducks; the abscissa represents the time point, and the ordinate Coordinates represent relative expression.

Fig. 2 is a map of the vector PiggyBac described in Example 2 of the present invention, wherein Xgene is BCL2L15.

Fig. 3 is the microscopic observation result of DF1 cells after transient transfection of BCL2L15 and negative control plasmids for 24 hours in Example 2 of the present invention.

Figure 4 is a Western blot verification of the overexpression effect of the BCL2L15 gene after the total cell protein was harvested after the transient transfection of BCL2L15 and the negative control plasmid in Example 2 of the present invention for 48 hours. The experimental treatment groups were BCL2L15 overexpression group (OE) and negative control group (Mock) in turn, and the GAPDH gene was used as an internal reference.

Fig. 5 is the overexpression of BCL2L15 gene in DF1 cells in Example 3 of the present invention, which inhibits the replication of influenza virus DK/49 (left) and GS/65 (right) in Example 6, and the abscissa indicates the collection of cell supernatant after infection The time point of the solution, the vertical axis represents the logarithmic value of EID50.

Fig. 6 shows the expression changes of endogenous BCL2L15 gene mRNA after DF1 cells were infected with influenza virus AIV (DK/49) for 48 hours in Example 3 of the present invention, with GAPDH gene as an internal reference.

7 shows the relative expression changes of different forms of virus RNA before and after cells overexpress BCL2L15 gene in Example 3 of the present invention and after infection with influenza virus AIV (DK/49), with GAPDH gene as an internal reference.

detailed description

Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Included in the experimental operation of this embodiment:

1) Extraction of total RNA and reverse transcription reaction

The extraction of total RNA from tissues or cells used Trizol reagent produced by Invitrogen Company, and was operated strictly according to the product instructions; the reverse transcription reaction was performed using MMLV reverse transcriptase reagent from Promega Company, and was operated strictly according to the instructions.

2) Gene cloning and vector construction

The cDNA obtained by reverse transcription was used as a template, and specific primers were used for PCR amplification. After the product was recovered and purified by gel, it was connected to the pEasy-Bluntingsimple vector, and a single clone colony was picked, and the plasmid was extracted after sequencing and identification. After double digestion of the plasmid, use T4 ligase to connect the target gene to the corresponding vector.

3) Cell culture and transfection

Chicken embryo fibroblast cell line (DF1) was cryopreserved by our laboratory earlier. The cell resuscitation culture was strictly aseptically operated, and the complete medium (DMEM+10% FBS) was added for culture, and the culture was carried out at 37°C and 5% CO ₂ , and the medium was changed every two days. Cell Transfection Reference HDTransfectionReagent (Promega) manual operation.

4) Western blot

Western blot was carried out in strict accordance with standard experimental methods. The primary antibody was Anti-flag antibody from Abcam, and the secondary antibody was HRP-labeled goat anti-mouse antibody from Beijing Zhongshan Jinqiao.

5) H5N1 subtype influenza virus challenge experiment

The GS65 and DK49 challenge experiments were completed in the P3 laboratory of Harbin Veterinary Research Institute. The virus growth curve was measured at 12h, 24h, 36h, 48h, 60h and 72h, a total of 6 time points were selected. The challenge dose was MOI=0.001, and the virus titer was determined The method is to measure the median infectious dose (EID50) of chicken embryos, and calculate the EID50 value according to the Reed-Muench method.

6) Using realtime RT-PCR technology to detect the expression of host factors and influenza virus genes

The fluorescence real-time quantitative PCR instrument is ABI7500 produced by ABI Company, and the quantitative reagent is from Roche, Germany. 480SYBRGreenIMaster, and operate in accordance with the product instructions. The original Ct value was converted into relative gene expression by 2- ^ΔΔCt method, and GAPDH was used as an internal reference gene.

Example 1 Obtaining the sequence of the full-length coding region of the duck BCL2L15 gene by means of molecular biology experiments

Referring to the duck genome reference sequence on the Ensemble website, and according to the transcriptome splicing sequence, design the duck BCL2L15 gene full-length CDS region cloning primer BF/BR, the sequence is as follows:

BF: 5'-CAATGACAACGTTTGAGGAACAGAC-3',

BR: 5'-GTAGTAAAACACTTCCTTCTCAGTCA-3'.

Using the cDNA of duck spleen tissue as a template, NEB company's Q5 high-fidelity polymerase was used for PCR amplification, and the amplified product was recovered by gel and ligated to T carrier for sequencing. After sequence comparison analysis, it was found that the full-length coding region of the duck BCL2L15 gene is 483bp (SEQ ID NO.1), encoding a total of 160 amino acids (SEQ ID NO.2).

In this example, the full-length coding region sequence of the duck BCL2L15 gene was successfully cloned.

Example 2 Transient overexpression of BCL2L15 gene in cells by cytology experiment method

1. Construction of duck BCL2L15 gene overexpression vector

According to the coding region sequence of duck BCL2L15 gene, the eukaryotic expression vector primer eBF/eBR was designed, and the upstream and downstream primers were respectively introduced into MluI and PmeI restriction sites (underlined); in addition, the upstream primer was before the start codon ATG Introduce the kozak (box marked) sequence, and introduce the flag tag (box marked) at the C-terminus of the target gene. The primer sequences are as follows:

eBF: 5'-CG ACGCGT ATGACAACGTTTGAGGAACAGACGA-3',

eBR: 5'-GG GTTTAAAC TCA GTCATCCAAGTTTCTCCCATCCTCCA-3'.

The CDS sequence of the amplified BCL2L15 gene (as shown in the sequence table CDS) was introduced into the original vector PiggyBac-Xgene (the vector map is shown in Figure 2), and the PiggyBac-BCL2L15 vector was constructed. Since the PiggyBac empty vector has a strong CAG promoter, it can efficiently and highly express the BCL2L15 gene introduced into it.

2. Transient overexpression of BCL2L15 gene in cells

The PiggyBac-BCL2L15 plasmid and the PiggyBac-Xgene empty vector plasmid not connected with the duck target gene CDS region sequence were transfected into the chicken DF1 cell line using FugeneHD (Promega). It was also set as a negative control group (DF1). After 24 hours of transfection, the cell state and transfection efficiency were observed under a microscope (as shown in FIG. 3 ).

3. Verification of duck BCL2L15 gene overexpression vector

The above cells transfected with PiggyBac-BCL2L15 and PiggyBac-Xgene empty vector were cultured in a CO ₂ incubator for 48 hours, the total protein of the cells was extracted, and the Anti-flag tag antibody was used for Western blot detection. The results of Western blot showed that the PiggyBac-BCL2L15-C-Flag vector could efficiently and highly express BCL2L15 protein in DF1 cells (as shown in Figure 4). The above transfection group of cells (overexpression of the target gene group, negative control NC and DF1 group) can be used for the next step of the challenge experiment.

Example 3 Verification of Duck BCL2L15 Gene Resistance to AIV Infection Using Cytological Experiments

1. Duck BCL2L15 gene inhibits influenza virus replication

Inoculate the transfected PiggyBac-BCL2L15, PiggyBac-Xgene empty vector (NC) and DF1 cell lines in good growth state in a 12-well cell culture plate at a density of 2×10 ⁵ cells/ml, and attack after the cells are stably adhered to the wall. poison. Two strains of H5N1 subtype avian influenza virus, namely DK/49 and GS/65, were selected for the challenge experiment, and the challenge dose was MOI=0.001, and the experiment was set to repeat independently in 3 wells. Cell supernatants were collected at 12h, 24h, 36h, 48h, 60h and 72h after challenge for EID50 detection. The cell challenge experiment was completed in the P3 laboratory of Harbin Veterinary Research Institute.

The challenge results showed that compared with the negative control group (NC), the duck BCL2L15 gene inhibited the replication of DK/49 influenza virus, and reached a significant difference at 36h (P<0.05), and reached a very significant difference at 48h and 72h (P<0.01 , Figure 5); meanwhile, duck BCL2L15 effectively inhibited the replication of GS/65 influenza virus, reaching a very significant difference (P<0.01) at 24h and 60h, and a significant difference at 36h and 48h (P<0.05, Figure 5). Among them, the negative control group (NC) and the DF1 group had the same trend in virus growth curve, and there was no significant difference.

2. The expression of BCL2L15 gene was significantly increased after the cells were infected with AIV

The present invention utilizes RealtimeRT-PCR technology to detect the relative expression of BCL2L15 gene mRNA in DF1 cells infected with AIV (DK/49) virus for 48 hours, and the results show that: after DF1 cells are infected with AIV virus, the expression level of endogenous BCL2L15 gene mRNA is significantly increased. Expression level (P<0.01, Figure 6).

3. Duck BCL2L15 gene significantly inhibits the expression of various forms of AIV virus RNA

The present invention further utilizes RealtimeRT-PCR technology to detect the expression changes of influenza virus M gene vRNA, cRNA and mRNA after the cells are infected with AIV (DK/49) virus before and after overexpressing the BCL2L15 gene for 48 hours. The results show that: after the cells are infected with AIV, Overexpression of the BCL2L15 gene significantly inhibited the expression of influenza virus M gene cRNA (P<0.05, Figure 7), significantly inhibited the expression of influenza virus M gene vRNA (P<0.01, Figure 7), and significantly inhibited the expression of influenza virus M gene vRNA (P<0.01, Figure 7). expression has no effect.

In summary, the inventors analyzed the effect of BCL2L15 gene on the replication of AIV virus in DF1 cells by comparing the virus content of AIV in DF1 cells overexpressing BCL2L15 gene and negative control (NC) cells. The final conclusion is that the duck BCL2L15 gene can inhibit the replication of AIV virus.

The gene encoding BCL2L15 protein described in the present invention can be used to prepare transgenic animals resistant to viruses such as AIV, provides a new method for broad-spectrum disease-resistant breeding of livestock and poultry, and has very broad application prospects.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1. The application in affecting virus replication of the 1.BCL2L15 gene, it is characterized in that, described BCL2L15 gene is the nucleotide sequence shown in SEQIDNO.1 or nucleotide sequence shown in SEQIDNo.1 is substituted, lacks and/or adds the nucleotide sequence derived by SEQIDNo.1 with equal function that one or several nucleotide obtains.
2. 2. application according to claim 1, it is characterised in that suppress virus duplication in cell by BCL2L15 gene process LAN in cell, or make the duplication of virus in cell dramatically increase by knocking out BCL2L15 gene.
3. 3. application according to claim 1 and 2, it is characterised in that described virus is bird flu virus.
4. 4. application according to claim 3, it is characterised in that by the CDS sequence construct of BCL2L15 gene in the carrier of efficiently expressing exogenous gene, recombinant vector can being obtained, and recombinant vector is imported in birds DF1 cell.
5. 5. application according to claim 4, it is characterised in that described carrier is the carrier for expression of eukaryon containing strong promoter.
6. 6. the transgenic cell containing BCL2L15 gene.
7. 7. transgenic cell according to claim 6, it is characterised in that described transgenic cell is chick embryo fibroblast immortalized cell line DF1.
8. 8. the vehicles cells for production of vaccine, it is characterised in that described vehicles cells is the cell having knocked out BCL2L15 gene.
9. 9. the construction method of transgenic animal, it is characterised in that described method includes proceeding to BCL2L15 gene in animal body.