CN110699487A - Antisense RNA for diagnosing avian leukosis virus tumorigenesis, preparation method, application and primer for constructing over-expression vector - Google Patents
Antisense RNA for diagnosing avian leukosis virus tumorigenesis, preparation method, application and primer for constructing over-expression vector Download PDFInfo
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Abstract
The invention relates to the field of animal virology, molecular biology and genetic engineering, in particular to antisense RNA for diagnosing tumorigenesis of avian leukosis virus, a preparation method, application and a primer for constructing an over-expression vector. The RNA is derived from long-chain non-coding RNA transcribed by a cyclin dependent kinase CDK6 gene antisense chain in chicken genome No. 2 chromosome, and is identified by RACE and PCR cloning technology and named AS chCDK 6-AS. Then, pcDNA3.1-chCDK6-AS overexpression vector was constructed. In vitro experiments show that the infection of avian leukosis virus subgroup J can obviously up-regulate the expression of chicken DF-1 cells and HD11 cells chCDK 6-AS. The invention not only provides a new means for diagnosing the tumorigenesis of the avian leukosis virus, but also provides a new idea for the research of the tumorigenic mechanism of the avian leukosis virus.
Description
Technical Field
The invention relates to the field of animal virology, molecular biology and genetic engineering, in particular to antisense RNA for diagnosing tumorigenesis of avian leukosis virus, a preparation method, application and a primer for constructing an over-expression vector.
Background
Cyclin-dependent kinase (CDKs) is an important family of protein kinases involved in tumorigenesis, which have been found to regulate the cell cycle, and functions related to transcriptional regulation and differentiation of nerve cells. Such enzymes are found in all eukaryotes known, and their regulation in the cell cycle is well conserved evolutionarily. CDKs are relatively small proteins with molecular weights between 34 and 40 kilodaltons, containing multiple kinase domains. Wherein CDK6 binds to cyclin DI, D2 and D3, it phosphorylates the product pRB of the retinoblastoma tumor suppressor gene. RB (retinoblastoma protein) is a nuclear phosphoprotein, which affects the cell cycle. At the end of cell G1, the non-phosphorylated RB protein is phosphorylated by a complex of cyclins and CDKs, thereby releasing the transcription factor E2F that binds specifically to RB, activating the cell into S phase.
Studies have shown that many viral infections can target CDKs, leading to abnormal cell cycle progression. The active kinase complex formed by functional cyclin D homologues encoded by the herpes viruses KSHV (Kaposi's sarcoma-associated herpesvirus) and HVS (herpesvirus saimiri) and CDK6 stimulates cell cycle progression in resting fibroblasts. In addition, CDK6/KSHV-cyclin complexes were not sensitive to inhibition by p16 and p27, and ectopic expression of KSHV-cyclins overcome cell cycle arrest caused by p 16. These mechanisms confer oncogenic properties to these viruses.
Antisense lncRNA refers to an RNA molecule that is transcribed from the antisense strand of a gene (usually a protein-encoding gene) and has some overlapping (overlapping) relationship with the mRNA of that gene. About 70% of the genes were found to have antisense lncRNA. More importantly, the antisense lncRNA can be widely involved in the expression regulation of protein coding genes, and the expression of mRNA and protein of a plurality of genes has correlation with the expression of the antisense lncRNA. The research finds that the mechanism of the antisense lncRNA for regulating the expression of the sense gene is diversified, the expression of exogenous p15AS in mouse embryonic stem cells causes p15 silence through heterochromatin formation and DNA methylation after embryonic stem cell differentiation, and the cell proliferation is accelerated. In contrast, BACE1-AS can improve the stability of the sense gene BACE1 mRNA, thereby up-regulating the expression of protein and participating in the pathophysiological process related to Alzheimer disease.
There has been little research on antisense RNA encoding genes in poultry. At present, a long-chain non-coding RNA transcribed by a chicken CDK6 antisense chain is found in a chicken genome through high-throughput sequencing, and the full length of the RNA is identified by using a rapid amplification at cDNA ends technology (RACE), which is named chCDK 6-AS. In recent years, poultry breeding industry develops rapidly, however, the incidence rate of neoplastic diseases is higher and higher, great loss is caused to the breeding industry, mixed infection sometimes occurs, and the common viral tumor diseases of chickens can be divided into three types: marek's disease, leukemia, and reticuloendotheliosis of chickens. Research shows that the exogenous J subgroup avian leukosis virus ALV-J infection can obviously increase CDK6 expression, promote cell proliferation and accelerate cell cycle progress, and finally cause tumorigenesis. Meanwhile, the experiment finds that ALV-J infection can also remarkably induce the expression of chCDK6-AS, so that the further understanding of the role played by chCDK6-AS in the virus infection and the tumor formation process and the expression rule thereof are particularly important.
Disclosure of Invention
The invention aims to provide an antisense RNA for diagnosing the tumorigenesis of avian leukosis virus, a preparation method, application and a primer for constructing an over-expression vector.
The technical scheme of the invention is as follows:
an antisense RNA for diagnosing the tumorigenesis of avian leukosis virus, which is a long non-coding RNA chCDK6-AS transcribed from the antisense strand of CDK6 gene, wherein the cDNA sequence of chCDK6-AS has the sequence shown in SEQ ID NO: 1.
Preferably, the RNA is used for diagnosing the tumorigenesis of the avian leukemia virus.
Preferably, the virus is a poultry tumor virus.
The preparation method of antisense RNA for diagnosing avian leukosis virus tumorigenesis comprises the following steps: 1) identifying the 5 'and 3' terminal sequences of chCDK 6-AS; 2) conventional PCR amplification of chCDK 6-AS; 3) TA clone sequencing to obtain a full-length sequence of chCDK 6;
the method is characterized in that in the step 1), universal primers UPM are respectively used for carrying out PCR amplification with 5 '-terminal or 3' -terminal gene specific primers, and the 5 '-terminal and 3' -terminal sequences of chCDK6-AS are obtained by clone sequencing, wherein the nucleotide sequences of the primers chCDK6-AS-5 '-RACE and chCDK 6-AS-3' -RACE are AS follows:
chCDK6-AS-5’-RACE：
5′-GCTAAGTGATTACAGTTTGAAACCG-3′(SEQ ID NO.6)；
chCDK6-AS-3’-RACE：
5′-CCTATTTGATTCAGATGTGTTCTCTCACTC-3′(SEQ ID NO.7)。
preferably, the nucleotide sequences of the primers chCDK6-AS-F and chCDK6-AS-R used in the step 2) are AS follows:
chCDK6-AS-F:5′-GAGCTCTGGTTCACGTCAG-3′(SEQ ID NO.2)
chCDK6-AS-R:5′-TATCCCTTTGTATTGCAAATTGTTAGA-3′(SEQ ID NO.3)。
preferably, the primers chCDK6-AS-F and chCDK6-AS-R are used for amplifying the full-length sequence of chCDK 6-AS.
The primer of the over-expression vector plasmid for constructing the antisense RNA for diagnosing the tumorigenesis of the avian leukosis virus is characterized by comprising the following primer sequences:
chCDK6-AS-EcoRI-F:
5′-ggaattcGAGCTCTGGTTCACGTCAG-3′
chCDK6-AS-XbaI-R:
5′-gctctagaTATCCCTTTGTATTGCAAATTGTTAGA -3′。
preferably, the application of the primers chCDK6-AS-EcoRI-F and chCDK6-AS-XbaI-R in the construction of pcDNA3.1-chCDK6-AS over-expression vector plasmid.
Preferably, the overexpression vector can remarkably enhance the expression of antisense RNA chCDK6-AS of the chicken CDK6 cell gene.
Preferably, the overexpression vector is applied to the research of an avian leukosis virus tumorigenic mechanism.
The invention provides a chicken CDK6 gene antisense RNA for diagnosing avian leukosis virus tumorigenesis, which is derived from long-chain non-coding RNA transcribed by a cyclin-dependent kinase CDK6 gene antisense chain in No. 2 chromosome of a chicken genome, and is named AS chCDK6-AS after being identified by RACE and PCR cloning technology. Then, pcDNA3.1-chCDK6-AS overexpression vector was constructed. In vitro experiments show that the infection of avian leukosis virus subgroup J can obviously up-regulate the expression of chicken DF-1 cells and HD11 cells chCDK 6-AS. The invention not only provides a new means for diagnosing the tumorigenesis of the avian leukosis virus, but also provides a new idea for the research of the tumorigenic mechanism of the avian leukosis virus.
Drawings
FIG. 1 is a chart of chCDK6-ASRACE agarose gel electrophoresis;
FIG. 2 is a schematic representation of the location of chCDK6-AS on chromosome 2 of chicken;
FIG. 3 is a graph showing the results of fluorescent quantitative PCR detection of chCDK6-AS after ALVJ virus infection of cells;
FIG. 4 is a map of over-expression vector pcDNA3.1-chCDK 6-AS;
FIG. 5 is a graph showing the results of fluorescent quantitative PCR detection after overexpression of chCDK 6-AS.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
Determination of full-Length sequence of chCDK6-AS
1. Identification of 5 'and 3' terminal sequences of chCDK6-AS
Supplied mainly by Takara
RACE 5 '/3' Kit.
First, by
Reagent extracts the total RNA of chicken macrophage line HD11, and then removes the genome with RNase-freeDNase I. Mu.g of the genome-depleted RNA was reverse transcribed to synthesize 5 '-or 3' -RACE products, respectively, by the action of SMARTScribeReverse Transcriptase (supplied from kit).
Then according to
The RACE 5 '/3' Kit was performed by PCR amplification using the universal primer UPM and the 5 '-end or 3' -end gene specific primer (gene-specific primer, GSP), respectively (see FIG. 1 for RACE agarose gel electrophoresis), and sequencing by cloning to obtain the 5 'end and 3' end sequences of chCDK 6-AS. Wherein the nucleotide sequences of the primers chCDK6-AS-5 '-RACE and chCDK 6-AS-3' -RACE are AS follows:
chCDK6-AS-5’-RACE：
5′-GCTAAGTGATTACAGTTTGAAACCG-3′(SEQ ID NO.6)
chCDK6-AS-3’-RACE：
5′-CCTATTTGATTCAGATGTGTTCTCTCACTC-3′(SEQ ID NO.7)。
2. conventional PCR amplified chCDK 6-AS. Wherein the nucleotide sequences of the primers chCDK6-AS-F and chCDK6-AS-R are AS follows:
chCDK6-AS-F:5′-GAGCTCTGGTTCACGTCAG-3′(SEQ ID NO.2)
chCDK6-AS-R:5′-TATCCCTTTGTATTGCAAATTGTTAGA-3′(SEQ ID NO.3)。
the reaction system comprises: 100ng of chicken macrophage line HD11 cDNA product, 2. mu.L (10. mu.M) of forward primer and 2. mu.L (10. mu.M) of reverse primer, 1. mu.L of Phanta Super-Fidelity DNA Polymerase, 10. mu.L of 5 XSF Buffer, 1. mu.L (10. mu.M) of dNTP Mix and 33. mu.L of ddH 2O.
The reaction conditions are as follows: 3min at 95 ℃; 35 cycles of 95 ℃ for 15s, 58 ℃ for 90s, 72 ℃ for 1 min; 7min at 72 ℃; maintaining the temperature at 4 ℃.
TA clonal sequencing the full-length sequence of chCDK6 (obtained by clonal sequencing is the cDNA sequence) was obtained, and the position of the sequence on chromosome 2 of chicken is schematically shown in FIG. 2. The specific cDNA sequence is shown in SEQ ID NO. 1:
GAGCTCTGGTTCACGTCAGCTGCTGGATGGCTGGAAATTTACCCCATTTGAACACTGCACAAGCTTTCAAAGGCCTTTGTTTAAGTCAAGGCAACTTCTCCACTAACTTTTACTCCAAGCAACATTTTCTGCCCGGAAGGCAAAAGGCTCACCCATGTGTGGTGCTGTTTACTTTCAGCTTCCACCGAACACAAAAAGAACTGATGGTGCTCACTTGGCCTTGTAGAATCATTTTTGAGAACAAGTTCCTATTTGATTCAGATGTGTTCTCTCACTCTCTGTATTAAAAACACAATGAAATACATAATTTTAAGGGATTTTTTAAAATGTAAAAGTGTATCGGTTTCAAACTGTAATCACTTAGCTAAAAGAAAGATTTGTAGCTGTTTTCTGCAAGAACTGAATGAGCTATTTTTGTGCATGCTATTCCTAAGTGTGTTTTATGGTTCTTATTTTGTGTATTTATTGCATATCAGTATGCAATGCATGCTCAGTTTTAGACAAAAGGGAAGAAATAACAATTGCCAAAAGTATGCTTGTCAGTTCGTATATCCACACCTTTCTTCTCTTAATGGCCTAGATCATTTAAATCAACAAAAAAGTTTCTTATTGCTTTTCAGAAGTTACTGGGATTTGATGGCTACAGAGTCTGGTCCAGAAACTGGAATTATATTACAGCCATTATCTAACAATTTGCAATACAAAGGGATA。
example 2
RT-qPCR detection of chCDK6-AS expression after ALVJ virus infection of DF-1 and HD11 cells
In this example 2, using the full-length chCDK6-AS sequence obtained in example 1, strand-specific reverse transcription primers and RT-qPCR primers were designed.
1. Cell recovery and culture
And taking out the frozen cells from the liquid nitrogen, placing the frozen cells in a water bath kettle at 37 ℃, shaking the frozen cells until the frozen cells are completely thawed, slowly sucking the cells into a centrifuge tube containing 2ml of MEM culture medium, centrifuging the cells at 1000g, and discarding the supernatant after 5 min. Suspending the precipitate in 10% culture medium, transferring to cell flask, and culturing DF-1 cells at 37 deg.C and 5% CO2Culturing in incubator, and culturing HD11 cells at 41 deg.C and 5% CO2Culturing in an incubator;
2. inoculating chicken DF-1 cells and HD11 cells to a 6-hole cell culture plate, standing overnight, infecting ALV-J virus after the cells are attached to the wall, and replacing a fresh culture medium after infecting for 12 h;
3. 48 hours after infection, cells were collected,
reagent extracts cellular Total RNA using PrimeScriptTMRT reagent Kit with gDNA Eraser Kit and specificity reverse transcription primer reverse transcription 1 μ gRNA, fluorescence quantitative PCR method detects chCDK6-AS expression;
by fluorescent quantitative PCR, we observed that ALVJ virus infection could significantly activate chCDK6-AS expression in DF-1 cells and HD11 cells (fig. 3).
Example 3
construction of chCDK6-AS overexpression vector and RT-qPCR detection of chCDK6-AS expression after overexpression
In this example 3, pcDNA3.1-chCDK6-AS overexpression plasmid was constructed using the full-length sequence of chCDK6-AS obtained in example 1.
1. Primers were designed based on the full-length sequence of chCDK6-AS, and EcoRI and Xbal cleavage sites were added upstream and downstream, respectively, and the nucleotide sequences were AS follows:
chCDK6-AS-EcoRI-F:
5′-ggaattcGAGCTCTGGTTCACGTCAG-3′(SEQ ID NO.4)
chCDK6-AS-XbaI-R:
5′-gctctagaTATCCCTTTGTATTGCAAATTGTTAGA-3′(SEQ ID NO.5)。
2. using chicken cDNA AS a template to amplify full length of chCDK6-AS by using high fidelity enzyme, wherein a reaction system comprises: 10 μ L of 5 XSSF Buffer, 1 μ L of dNTP Mix, 2 μ L of forward primer, 2 μ L of reverse primer, 2 μ L of cDNA template, 1 μ L of Phanta Super-Fidelity DNA Polymerase and 32 μ L of ddH 2O. The reaction conditions are as follows: 3min at 95 ℃; 10s at 95 ℃, 30s at 56 ℃, 45s at 72 ℃, 40 cycles, 7min at 72 ℃; cutting the gel and recovering the target fragment. Carrying out enzyme digestion on the pcDNA3.1(+) vector by EcoR I and Xbal, recovering the chCDK6-AS fragment, and connecting the target fragment and the pcDNA3.1 (+);
3. the ligation product is transformed into DH5 alpha competent cell, single clone is selected for sequencing, plasmid is extracted for correct sequencing, and then the plasmid is named pcDNA3.1-chCDK6-AS, and the plasmid map is shown in figure 4;
4. the day before transfection, DF-1 cells were plated in 12 well cell culture dishes and placed in 1mL antibiotic-free medium to achieve 70-90% confluence at the time of transfection. 1.6. mu.g of plasmid was transfected into DF-1 cells using Lipofectamine 2000 transfection reagent, and the medium was changed after 6 hours;
5. after 48h of transfection, the cells were collected,
reagent extracts cellular Total RNA using PrimeScriptTMRTreagent Kit with gDNA Eraser Kit and specific reverse transcription primer reverse transcription 1 u gRNA, fluorescence quantitative PCR method detects chCDK6-AS expression.
The results of the strand-specific fluorescent quantitative PCR assay are shown in FIG. 5: 48h after DF-1 cells were transfected, the expression level of chCDK6-AS was increased by about 160 times.
Sequence listing
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cctatttgat tcagatgtgt tctctcactc 30
Claims (10)
1. An antisense RNA for diagnosing the tumorigenesis of avian leukosis virus, which is a long-chain non-coding RNA chCDK6-AS transcribed from the antisense strand of CDK6 gene, wherein the cDNA sequence of chCDK6-AS has the sequence shown in SEQ ID NO: 1.
2. The use of an antisense RNA for the diagnosis of tumorigenesis of avian leukemia virus according to claim 1, wherein the RNA is used for the diagnosis of tumorigenesis of avian leukemia virus.
3. The use of an antisense RNA for the diagnosis of tumorigenesis of avian leukemia virus according to claim 2, wherein the virus is a poultry tumor virus.
4. The method for preparing an antisense RNA for diagnosing tumorigenesis of avian leukemia virus according to claim 1, comprising the steps of: 1) identifying the 5 'and 3' terminal sequences of chCDK 6-AS; 2) conventional PCR amplification of chCDK 6-AS; 3) TA clone sequencing to obtain a full-length sequence of chCDK 6;
the method is characterized in that in the step 1), universal primers UPM are respectively used for carrying out PCR amplification with 5 '-terminal or 3' -terminal gene specific primers, and the 5 '-terminal and 3' -terminal sequences of chCDK6-AS are obtained by clone sequencing, wherein the nucleotide sequences of the primers chCDK6-AS-5 '-RACE and chCDK 6-AS-3' -RACE are AS follows:
chCDK6-AS-5’-RACE：
5′-GCTAAGTGATTACAGTTTGAAACCG-3′(SEQ ID NO.6)；
chCDK6-AS-3’-RACE：
5′-CCTATTTGATTCAGATGTGTTCTCTCACTC-3′(SEQ ID NO.7)。
5. the method for preparing an antisense RNA for diagnosing tumorigenesis of avian leukemia virus according to claim 4, wherein the nucleotide sequences of the primers chCDK6-AS-F and chCDK6-AS-R used in step 2) are AS follows:
chCDK6-AS-F:5′-GAGCTCTGGTTCACGTCAG-3′(SEQ ID NO.2)
chCDK6-AS-R:5′-TATCCCTTTGTATTGCAAATTGTTAGA-3′(SEQ ID NO.3)。
6. the method for preparing an antisense RNA for diagnosing tumorigenesis of avian leukemia virus according to claim 5, wherein the primers chCDK6-AS-F and chCDK6-AS-R are used for amplifying the full-length sequence of chCDK 6-AS.
7. The primer for constructing the antisense RNA overexpression vector plasmid for diagnosing tumorigenesis of avian leukemia virus according to claim 1, wherein the primer has the following sequence:
chCDK6-AS-EcoRI-F:
5′-ggaattcGAGCTCTGGTTCACGTCAG-3′
chCDK6-AS-XbaI-R:
5′-gctctagaTATCCCTTTGTATTGCAAATTGTTAGA-3′。
8. the primers for constructing an antisense RNA over-expression vector plasmid for diagnosing tumorigenesis of avian leukemia virus according to claim 7, wherein the primers chCDK6-AS-EcoRI-F and chCDK6-AS-XbaI-R are used for constructing pcDNA3.1-chCDK6-AS over-expression vector plasmid.
9. The primers for constructing the plasmid of the overexpression vector of the antisense RNA for diagnosing the tumorigenesis of the avian leukemia virus according to claim 7, wherein the overexpression vector can significantly enhance the expression of the antisense RNA chCDK6-AS of the chicken CDK6 gene.
10. The primer for constructing the plasmid of the overexpression vector of the antisense RNA for diagnosing tumorigenesis of avian leukemia virus according to claim 9, wherein the overexpression vector is used for the research of tumorigenic mechanism of avian leukemia virus.
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| CN111534517A (en) * | 2020-05-16 | 2020-08-14 | 扬州大学 | Antisense RNA MYC-AS1 for inhibiting expression of protooncogene c-MYC and application thereof |
| CN111575288A (en) * | 2020-05-16 | 2020-08-25 | 扬州大学 | ch-MYC-AS1 for inhibiting chicken c-MYC gene expression and J subgroup avian leukosis virus proliferation and application thereof |
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