CN105567701B - DCSTAMP gene and its application - Google Patents

Abstract

The present invention provides a kind of DCSTAMP gene of duck and its amino acid sequences of coding, duck DCSTAMP gene provided by the present invention can be used for inhibiting influenza virus, the duplication of AIV virus can especially be significantly inhibited, therefore deep functional study can be carried out for duck DCSTAMP gene, so that it is determined that the key protein structural domain or amino acid of its anti-AIV virus.To provide new approach using gene editings method, the transgenosis agricultural animal excellent variety for cultivating the multiple types such as anti-AIV virus such as transgenic technologys.

Description

DCSTAMP gene and its application

technical field

The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to a DCSTAMP gene and its application.

Background technique

Influenza viruses are minus-strand RNA viruses containing eight RNA genome segments. Highly Pathogenic Avian Influenza (HPAI) is a severe avian-based infectious disease caused by influenza A virus of the family Orthomyxoviridae.

Waterfowl, including domestic ducks, are natural hosts of influenza virus, and all 16 HA and 9 NA subtypes in different combinations can be isolated in waterfowl. Influenza virus has always maintained the characteristics of low pathogenicity to waterfowl. Some strains with low pathogenicity to waterfowl are highly pathogenic to chickens or other hosts. However, with the continuous evolution of influenza virus, the balance between waterfowl and influenza virus has been broken. For example, in 2002, the H5N1 subtype strain that killed waterfowl appeared in Hong Kong for the first time, and in 2005, a large-scale outbreak of H5N1 subtype influenza virus in Qinghai Lake killed migratory birds. There are many ways for new strains circulating in the human population: genetic rearrangement of avian influenza virus or other influenza virus with human influenza virus to produce circulating strains that infect humans, circulating strains produced by avian influenza viruses after adaptation in pigs, Avian influenza virus spreads to humans and produces circulating strains and old strains of human influenza virus re-populate after a lapse of several years. In addition, there are antigenic drift of human influenza virus itself.

In a word, the biggest feature of influenza A virus is that it has a wide range of hosts, many subtypes, various forms of variation, sudden onset, strong epidemicity 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 committed to identifying new immune genes against influenza viruses, and analyzing the host's impact on influenza virus infectivity and pathogenicity. And the molecular mechanism research of immune response, in order to improve the host's disease resistance and promote the development of new means of prevention and control of influenza virus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel gene related to resistance to influenza virus, and to provide the application of the gene in resistance to influenza virus.

The inventors of the present invention have found that waterfowl including domestic ducks are the storage hosts of the H5N1 subtype influenza virus in the research, and the virus does not kill waterfowl in the early stage, and the waterfowl itself does not show obvious clinical symptoms. With the continuous evolution of the virus, Strains with high virulence against waterfowl also appeared. Therefore, the inventors searched for genes related to resistance or susceptibility by comparing the differential gene expression of Peking ducks infected with two subtypes of H5N1 strains. Thus found and provided a duck DCSTAMP gene, the duck DCSTAMP gene has:

a) the nucleotide sequence shown in SEQ ID NO.1; or

b) The nucleotide sequence derived from a) with the equivalent function obtained by substitution, deletion and/or addition of one or several nucleotides to the nucleotide sequence shown in SEQ ID No. 1.

The present invention also provides the amino acid sequence encoded by the duck DCSTAMP gene.

The present invention also provides a vector containing the duck DCSTAMP gene.

Preferably, the vector is a PiggyBac-DCSTAMP vector obtained by introducing the CDS sequence of the duck DCSTAMP gene.

The present invention also provides a transgenic cell containing the duck DCSTAMP gene.

The transgenic cells can be avian cells or mammalian cells.

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

The present invention also provides the application of the duck DCSTAMP gene in influencing the replication of influenza virus.

Optionally, the influenza virus includes H5N1 type AIV virus, such as low pathogenic strain GS/65 (A/goose/Hubei/65/05) and high pathogenic strain DK/49 ( A/duck/Hubei/49/05).

Optionally, the application includes inhibiting the replication of AIV virus in cells by overexpression of the duck DCSTAMP gene in the cells, and significantly increasing the replication of AIV virus in cells knocked out of DCSTAMP.

The present invention also provides a method for constructing a transgenic animal, which comprises transferring the duck DCSTAMP gene of the present invention into animal cells.

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

The DCSTAMP gene provided by the present invention can be used to inhibit influenza virus, especially the replication of AIV virus can be significantly inhibited. Therefore, in-depth functional research can be carried out on the duck DCSTAMP gene to determine its key protein domains or amino acids against AIV virus. Using gene editing methods such as transgenic technology, we can obtain transgenic livestock and poultry with high inducible expression of DCSTAMP 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 mRNA sequence of duck DCSTAMP gene obtained by transcriptome data analysis in Example 1 of the present invention.

Figure 2 is a map of the vector PiggyBac described in Example 3 of the present invention, wherein the X gene is DCSTAMP.

Figure 3 shows the microscopic observation results of DF1 cells after transient transfection of DCSTAMP and negative control plasmids for 24 hours in Examples 4 and 5 of the present invention.

Figure 4 shows the Western blot verification of the overexpression effect of DCSTAMP gene after the total cell protein was collected after transiently transfecting DCSTAMP and negative control plasmids for 48 hours in Examples 4 and 5 of the present invention.

Figure 5 shows the overexpression of DCSTAMP gene in DF1 cells in Examples 4 and 5 of the present invention, which inhibits the replication of influenza virus DK/49 (left) and GS/65 (right) in Example 6.

Figure 6 shows the expression changes of endogenous DCSTAMP gene mRNA after DF1 cells were infected with influenza virus AIV (DK/49) for 48 hours in Examples 4 and 5 of the present invention, with GAPDH gene as an internal reference.

Figure 7 shows the relative expression changes of different forms of virus RNA before and after the cells overexpressed DCSTAMP gene in Examples 4 and 5 of the present invention and after infection with influenza virus AIV (DK/49), using GAPDH gene as an internal reference.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (New York: Gold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions suggested in the manufacturer's instructions.

Example 1

A novel anti-AIV infection gene, DCSTAMP, was discovered by analyzing transcriptome data.

Waterfowl, including domestic ducks, are the natural hosts of H5N1 subtype influenza virus, and after infection with some H5N1 subtype viruses, they do not show obvious clinical symptoms. As the virus continues to evolve, strains that are highly pathogenic to waterfowl also appear. In this study, two H5N1 subtype strains (namely, highly pathogenic A/duck/Hubei/49/05 (DK/49) and low pathogenic A/goose/Hubei/65/05 (GS/65) were selected. ) H5N1 virus), infected 4-week-old Shaoxing ducks. Through high-throughput sequencing, the gene expression profiles of spleen, brain and lung tissue of ducks infected with DK/49 or GS/65H5N1 influenza virus at 1, 2 and 3 days and control ducks were constructed respectively, and the gene expression profiles of the spleen, brain and lung tissues of ducks in the control group were respectively constructed to identify the genes involved in the body's anti-influenza virus. Influenza virus-related immune genes provide new methods for the treatment and prevention of avian influenza.

In the early stage, 4-week-old Shaoxing ducks were infected with DK/49 or GS/65H5N1 virus. After 1, 2, and 3 days after infection with AIV, spleen, brain and lung tissue samples were taken, total RNA was extracted, and a library was constructed for high-throughput sequencing. , by bioinformatics analysis. The analysis found that compared with the control group, the expression of DCSTAMP was significantly increased in the spleen, brain and lung tissues at 1, 2 and 3 days after infection with DK/49 or GS/65H5N1 virus (Figure 1). As shown, in two H5N1 strains (A/duck/Hubei/49/05, DK/49, high pathogenicity) and (A/goose/Hubei/65/05, GS/65, low pathogenicity), respectively, Expression patterns in spleen, lung, and brain tissues on days 1, 2, and 3 after infection with 4-week-old Shaoxing ducks; the abscissa represents time points, and the ordinate represents relative expression levels.).

Example 2

Obtaining the full-length coding region sequence of duck DCSTAMP gene by molecular biology method

Referring to the duck genome reference sequence on the Ensemble website, and according to the transcriptome splicing sequence, design the full-length CDS region cloning primer TF/TR of the duck DCSTAMP gene. The sequence is as follows:

TF: 5'-ATGCAAGCACTTGTCTCAACAGCCC-3',

TR: 5'-TCCAAAACAC TGCTGACCAT TAGCC-3'.

Using duck spleen tissue cDNA as a template, PCR amplification was performed using NEB's Q5 high-fidelity polymerase, and the amplified product was recovered by gel and then connected to T vector for sequencing. After sequence alignment and analysis, it was found that the full-length coding region of the duck DCSTAMP gene was 1431 bp (SEQ ID NO.1), encoding a total of 476 amino acids (SEQ ID NO.2).

The inventors successfully cloned the full-length coding region sequence of duck DCSTAMP gene.

Example 3

Transient overexpression of DCSTAMP gene in cells by cytological assay

1. Construction of duck DCSTAMP gene overexpression vector

According to the sequence of the coding region of duck DCSTAMP gene, the primers eTF/eTR of eukaryotic expression vector were designed, and the upstream and downstream primers were introduced into Mlu I and Pme I restriction sites (underlined) respectively; A kozak (bold) sequence was introduced before ATG, and a flag tag (bold) was introduced at the C-terminus of the target gene. The primer sequences are as follows:

eTF:

5'-CG ACGCGT GCCACCATGCAAGCACTTGTCTCAACAGCCCAGAATGC-3',

eTR:

5'-GG GTTTAAAC CTACTTATCGTCGTCATCCTTGTAATCCACCACATTGTCATTTACCATTGTC-3'.

The amplified CDS sequence of the DCSTAMP gene (as shown in the sequence table CDS) was introduced into the original vector PiggyBac-X gene (constructed by the inventor's laboratory in the early stage, the vector map is shown in Figure 2), and the PiggyBac-DCSTAMP was constructed. vector. Since the PiggyBac empty vector has a strong CAG promoter, the DCSTAMP gene introduced into it can be efficiently and highly expressed.

2. Transient overexpression of DCSTAMP gene in cells

The PiggyBac-DCSTAMP plasmid and the PiggyBac-X gene empty vector plasmid without the CDS region sequence of the duck target gene were used to transfect the chicken DF1 cell line with Fugene HD (Promega). The treatment group was also set as the negative control group (DF1). 24h after transfection, the cell state and transfection efficiency were observed under a microscope (as shown in Figure 3).

3. Validation of duck DCSTAMP gene overexpression vector

The cells transfected with the PiggyBac-DCSTAMP and PiggyBac-X gene empty vectors were cultured in a CO ₂ incubator for 48 h, and the total protein of the cells was extracted and detected by Western blot using Anti-flag-labeled antibody. Westernblot results showed that the PiggyBac-DCSTAMP-C-Flag vector could effectively and highly express DCSTAMP protein in DF1 cells (as shown in Figure 4, the experimental treatment groups were the overexpression DCSTAMP group (OE) and the negative control group (Mock) in turn. GAPDH gene was used as an internal control). The cells of the above transfection group (overexpressing the target genome, negative control NC and DF1 group) can be used for the next challenge experiment.

Example 4 Validation of duck DCSTAMP gene against AIV infection by cytological experimental method

1. Duck DCSTAMP gene inhibits influenza virus replication

Transfected PiggyBac-DCSTAMP-C-Flag vector (DCSTAMP), PiggyBac-Xgene empty vector (NC) and DF1 cell lines with good growth status were seeded in 12-well cell culture plates at a density of 2×10 ⁵ cells/ml. The challenge was performed after the cells were stably attached. Two strains of avian influenza viruses of H5N1 subtype were selected for the challenge experiment, namely DK/49 and GS/65, and the challenge dose was MOI=0.001. The experiment was repeated independently in 3 wells. 12h, 24h, 36h, 48h, 60h and 72h post-challenge cell supernatants were harvested for EID50 detection. Cell challenge experiments were completed in the P3 laboratory of Harbin Veterinary Research Institute.

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

In Fig. 5, the abscissa represents the time point when the cell supernatant was collected after infection, and the ordinate represents the logarithmic value of EID50.

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

The present invention uses Real time RT-PCR technology to detect the relative expression of DCSTAMP gene mRNA of DF1 cells 48 hours after infection with AIV (DK/49) virus, and the results show that: after DF1 cells are infected with AIV virus, the endogenous DCSTAMP gene mRNA significantly increases expression level (P<0.05, Figure 6).

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

The present invention further utilizes Real time RT-PCR technology to detect the expression changes of influenza virus M gene vRNA, cRNA and mRNA before and after the overexpression of DCSTAMP gene in cells and 48 hours after infection with AIV (DK/49) virus. The results show that: cells are infected with AIV After that, overexpression of DCSTAMP gene significantly inhibited the expression of influenza virus M gene vRNA and cRNA (P<0.01, Figure 7), but had no effect on influenza virus M gene mRNA expression.

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

The gene encoding the DCSTAMP protein provided by the 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.

The experimental operation of this embodiment includes:

1) Extraction of total RNA and reverse transcription reaction

The extraction of total RNA from tissues or cells uses Trizol reagent produced by Invitrogen Company, and the operation is carried out in strict accordance with the product instructions; the reverse transcription reaction uses MMLV reverse transcriptase reagent of Promega Company, and the operation is carried out in strict accordance with the instructions.

2) Gene cloning and vector construction

Using the cDNA obtained by reverse transcription as a template, PCR amplification was performed using specific primers. The product was recovered and purified by gel and then connected to the pEasy-Blunting simple vector, and single clone colonies were picked, and the plasmid was extracted after sequencing and identification were correct. After the plasmid was double digested, the target gene was ligated to the corresponding vector using T4 ligase.

3) Cell culture and transfection

The chicken embryo fibroblast cell line (DF1) was cryopreserved in our laboratory. Cell recovery and culture were performed strictly aseptically, and complete medium (DMEM+10% FBS) was added for culture at 37°C under 5% CO2 conditions, and the medium was changed every two days. Cell transfection reference HD Transfection Reagent (Promega) manual operation.

4) Western blot

Western blot was performed in strict accordance with standard experimental methods. The primary antibody was Anti-flag labeled antibody from Abcam, and the secondary antibody was HRP-labeled goat anti-mouse antibody from Beijing Zhongshan Golden Bridge. The primary antibody for GAPDH was purchased from Biyuntian Institute of Biotechnology.

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 determined at 6 time points, 12h, 24h, 36h, 48h, 60h and 72h. The challenge dose was MOI=0.001, and the virus titer was determined. The method was the determination of the half-infectious dose of chicken embryos (EID50), and the EID50 value was calculated according to the Reed-Muench method.

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

The fluorescence real-time quantitative PCR instrument was ABI 7500 produced by ABI Company, and the quantitative reagents were from Roche, Germany. 480 SYBR Green I Master and follow the product instructions. The original Ct value was converted into relative gene expression using the 2- ^ΔΔCt method, and GAPDH was used as the internal reference gene.

Although the present invention has been described in detail above with general description and specific embodiments, 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, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (3)

1. duck DCSTAMP gene is influencing non-diagnostic and/or non-treatment purpose the application in H5N1 type AIV virus replication, It is characterized in that, the nucleotide sequence of the duck DCSTAMP gene is as shown in SEQ ID NO.1.

2. application according to claim 1, which is characterized in that the H5N1 type AIV influenza virus be strain GS/65 or DK/49。

3. duck DCSTAMP gene answering in the transgenic chicken embryo fibroblast DF1 for preparing anti-H5N1 type AIV influenza virus With, which is characterized in that the application is that duck DCSTAMP gene, the duck are transferred in the chicken embryo fibroblasts system DF1 The nucleotide sequence of DCSTAMP gene is as shown in SEQ ID NO.1.