Immortalized goose ovary granular cell line and construction method thereof

Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to an immortalized goose ovary granular cell line and a construction method thereof. According to the present invention, the DNA sequence for coding SV40T and HPV viruses is introduced into the goose primary ovarian granular cells through the lentiviral vector, such that the immortalized cell line capable of stably expressing SV40T and HPV viruses and maintaining the same biological characteristics as the primary ovarian granular cells is constructed, and the stable proliferation of the immortalized cell line is achieved; the cell line construction method has relatively high repeatability and stability, can be applied to immortalization research of different varieties of goose ovarian granular cells, and is beneficial to promoting the development of goose breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and particularly relates to an immortalized goose ovary granulosa cell line and a construction method thereof. Background Art

[0002] Granulosa cells are a key cell type in the goose ovary. These cells surround the oocyte and form the outer structure of the follicle. They are essential for the growth, maturation and ovulation of the follicle and play a core role in follicle development and the production of reproductive hormones. Ovarian granulosa cells play a key role in the reproductive process of geese. Studying their function and regulatory mechanism is of great significance for improving the reproductive performance of geese.

[0003] The research methods at home and abroad mainly extract primary ovarian granulosa cells from living geese, but primary cells have a limited lifespan and poor proliferation ability when cultured in vitro, which makes it difficult to meet the needs of long-term in-depth research. Although some people have constructed immortalized goose ovarian granulosa cell lines by introducing SV40 genes in the prior art, the proliferation rate of immortalized cells constructed by introducing SV40 genes alone is slow.

[0004] Therefore, it is urgent to construct an immortalized goose ovary granulosa cell line with strong proliferation ability. Summary of the invention

[0005] The purpose of the present invention is to provide an immortalized goose ovary granulosa cell line. The immortalized goose ovary granulosa cell line provided by the present invention not only has a strong proliferation ability, but also has high repeatability and stability.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an immortalized goose ovarian granulosa cell line, which is obtained by transfecting a lentiviral vector carrying DNA sequences encoding SV40T and HPV viruses into primary goose ovarian granulosa cells;

[0008] According to the accession number of the DNA sequence encoding the SV40T virus in GenBank, which is NC_001669.1, the primers of the DNA sequence of the SV40T virus are shown in the nucleotide sequence table SEQ ID NO.1-2; the DNA sequence of the SV40T virus is 2075 bp;

[0009] According to the accession number of the DNA sequence encoding the HPV virus in GenBank, which is NC_001526.4, the primers of the DNA sequence of the HPV virus are shown in the nucleotide sequence table SEQ ID NO.3-4; the DNA sequence of the HPV virus is 729 bp.

[0010] Preferably, the lentiviral vector is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro.

[0011] The present invention also provides a method for constructing an immortalized goose ovary granulosa cell line, comprising using the above-mentioned lentiviral vector to mediate the DNA sequence encoding SV40T and HPV virus, and transfecting it into a host cell, and then screening it with puromycin; the host cell is a goose ovary granulosa cell;

[0012] According to the accession number of the DNA sequence encoding the SV40T virus in GenBank, which is NC_001669.1, the primers of the DNA sequence of the SV40T virus are shown in the nucleotide sequence table SEQ ID NO.1-2; the DNA sequence of the SV40T virus is 2075 bp;

[0013] According to the accession number of the DNA sequence encoding the HPV virus in GenBank, which is NC_001526.4, the primers of the DNA sequence of the HPV virus are shown in the nucleotide sequence table SEQ ID NO.3-4; the DNA sequence of the HPV virus is 729 bp.

[0014] The present invention also provides a process for constructing an immortalized goose ovary granulosa cell line, which specifically comprises the following steps:

[0015] S1, constructing the DNA sequences encoding SV40T and HPV viruses into the above-mentioned lentiviral vector to obtain a recombinant lentiviral vector;

[0016] S2, co-transfecting the recombinant lentiviral vector described in S1 with the lentiviral packaging helper plasmid into 293T cells for pseudovirus packaging;

[0017] S3. The pseudovirus of S2 was transfected into primary isolated goose ovary granulosa cells to obtain an immortalized cell line.

[0018] Preferably, the lentiviral vector is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro; and the lentiviral packaging helper plasmids are pMD2.G and psPAX2.

[0019] Preferably, the molar ratio of the recombinant lentiviral vector and the lentiviral packaging helper plasmid is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro-HPV-SV40T:pMD2.G:psPAX2=4:1:3.

[0020] Preferably, in addition to the above steps, the process of constructing the immortalized goose ovary granulosa cell line further comprises the step of measuring the cell proliferation activity of the obtained immortalized cell line.

[0021] The present invention also provides the use of the cell line or the cell line constructed by the construction method in goose disease research, wherein the goose is a geese with open eyes.

[0022] Beneficial effects of the present invention:

[0023] The present invention successfully constructed an immortalized cell line of goose ovarian granulosa cells with good proliferation effect using lentiviral vectors for the first time. The cell line construction method has high repeatability and stability, and is expected to be applied to the immortalization research of goose ovarian granulosa cells of different varieties, which will help promote the development of goose breeding industry. It is expected to provide stable cell line materials and reliable technical platforms for related research in the field of goose reproductive biology, filling the technical gap in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. It is obvious that the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 This is a cell status diagram of the immortalized cell line constructed using SV40T and HPV genes after culturing for 48 hours (40X);

[0026] Figure 2 This is a cell status diagram of the immortalized cell line constructed using SV40T and HPV genes after 96 hours of culture (40X);

[0027] Figure 3 This is a cell status diagram of the immortalized cell line constructed using the SV40 gene after culturing for 48 hours (40X);

[0028] Figure 4 This is a diagram of the cell status of the immortalized cell line constructed using the SV40 gene after 96 hours of culture (40X). DETAILED DESCRIPTION

[0029] The invention provides an immortalized goose ovary granulosa cell line and a construction method thereof.

[0030] The present invention provides an immortalized goose ovary granulosa cell line, which is an immortalized cell line obtained by transfecting goose ovary granulosa cells with SV40T and HPV viruses mediated by lentiviral vectors. The immortalized goose ovary granulosa cell line can be used as an in vitro model for goose ovary function research.

[0031] The present invention provides an immortalized goose ovary granulosa cell line with strong passage stability. The present invention provides a method for constructing an immortalized goose ovary granulosa cell line, using goose ovary granulosa cells as host cells, using lentiviral vectors to mediate SV40T and HPV virus DNA sequences to transfect the host cells, and then screening with puromycin to obtain the immortalized goose ovary granulosa cell line.

[0032] According to the accession number of the DNA sequence encoding SV40T virus in GenBank, which is NC_001669.1, the primers of the DNA sequence of SV40T virus are shown in the nucleotide sequence table SEQ ID NO.1-2; the DNA sequence of the SV40T virus is 2075bp; according to the accession number of the DNA sequence encoding HPV virus in GenBank, which is NC_001526.4, the primers of the DNA sequence of HPV virus are shown in the nucleotide sequence table SEQ ID NO.3-4; the DNA sequence of the HPV virus is 729bp.

[0033] The primers for SV40T and HPV are as follows: (Note: lowercase letters represent homology arms, and uppercase letters represent target gene fragments)

[0034] SV40T-F:cctggatacccgtattctagaATGGATAAAGTTTTAAACAGAGAGGAA T(SEQ IDNO.1)

[0035] SV40T-R:gcatTGTTTCAGGTTCAGGGGGGAGG(SEQ ID NO.2)

[0036] HPV-F:ccctgaacctgaaacaATGCACCAAAAGAGAACTGCAA(SEQ ID NO.3)

[0037] HPV-R: atccttcgcggccgcggatccTGGTTTCTGAGAACAGATGGGG (SEQ ID NO.4)

[0038] Furthermore, the method for constructing an immortalized goose ovary granulosa cell line comprises the following steps:

[0039] (1) constructing DNA sequences encoding SV40T and HPV viruses into a lentiviral vector to obtain a recombinant lentiviral vector;

[0040] (2) co-transfecting the recombinant lentiviral vector of (1) with a lentiviral packaging helper plasmid into 293T cells for pseudovirus packaging;

[0041] (3) The pseudovirus of (2) was transfected into primary isolated goose ovary granulosa cells to obtain an immortalized cell line.

[0042] Furthermore, the recombinant lentiviral vector is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro-HPV-SV40T, and the lentiviral packaging auxiliary plasmids are pMD2.G and psPAX2. The above vectors and plasmids can be purchased from the market.

[0043] More preferably, the molar ratio of the recombinant lentiviral vector to the lentiviral packaging helper plasmid is 4:1:3.

[0044] The aforementioned method further comprises the step of measuring the cell proliferation activity of the obtained immortalized cell line.

[0045] The goose breed of the present invention is the excellent local breed of open-eyed goose in Liaoning Province.

[0046] The present invention establishes an immortalized goose ovary granulosa cell line, which retains the characteristics of primary granulosa cells and can be used for related research on goose ovary function, filling the gap of no available cell line in the current research on goose ovary-related cell proliferation, differentiation, signal transduction and hormone secretion.

[0047] Lentiviral vectors are a viral vector system based on the human immunodeficiency virus (HIV). They can efficiently introduce target genes into primary cells or cell lines of animals and humans, and have the ability to infect both dividing cells and non-dividing cells.

[0048] Packaging plasmids are key components used in lentiviral packaging systems. They carry genes encoding key proteins required for lentiviral replication and assembly.

[0049] The process of transfecting cells refers to the technology of introducing exogenous nucleic acids (such as DNA or RNA) into eukaryotic cells in order to express specific genes or regulate gene expression in the cells. This process is crucial in the fields of gene function research, protein expression, gene therapy, etc.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0052] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0053] Example 1 Isolation and culture of primary goose ovary granulosa cells

[0054] Ovarian tissues of healthy adult geese were selected and cut into small pieces under sterile conditions. The tissue pieces were placed in a digestion solution containing 0.1% type II collagenase and digested at 37°C for 5 min. Cells were then collected by centrifugation and washed with pre-cooled PBS. The cells were inoculated in M199 medium containing 15% fetal bovine serum and placed at 38.5°C and 5% CO. 2 After 48 h of culture, the culture medium was replaced to remove non-adherent cells, and the cells were cultured until they reached 80% to 90% confluence for subsequent gene transfection experiments.

[0055] Example 2 Construction of immortalized goose ovary granulosa cell line

[0056] According to the DNA sequences of SV40T and HPV genes in the NCBI database, the corresponding specific primers were designed. DNA containing SV40T and HPV genes was used as a template for PCR amplification, followed by agarose gel electrophoresis. After the electrophoresis, the gel was cut to recover the target band and the product was purified. The lentiviral vector pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro was linearized and connected to the purified product by homologous recombination. The connection product was then transformed into Stbl3 competent cells, positive clones were screened, and the plasmid connected to the target gene was extracted.

[0057] The primers for SV40T and HPV are as follows: (Note: lowercase letters represent homology arms, and uppercase letters represent target gene fragments)

[0058] SV40T-F:cctggatacccgtattctagaATGGATAAAGTTTTAAACAGAGAGGAA T(SEQ IDNO.1)

[0059] SV40T-R:gcatTGTTTCAGGTTCAGGGGGGAGG(SEQ ID NO.2)

[0060] HPV-F:ccctgaacctgaaacaATGCACCAAAAGAGAACTGCAA(SEQ ID NO.3)

[0061] HPV-R: atccttcgcggccgcggatccTGGTTTCTGAGAACAGATGGGG (SEQ ID NO.4)

[0062] The lentiviral vector pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro carrying SV40T and HPV genes was constructed and co-transfected with packaging plasmids pMD2.G and psPAX2 into 293T cells. The viral supernatant was collected and the virus was concentrated by ultracentrifugation.

[0063] Primary goose granulosa cells were inoculated in a 6-well plate. When the cells grew to a confluence of 50% to 60%, concentrated lentivirus solution with an MOI of 50 was added, and 8 μg / mL of polybrene was added. After culturing for 6 hours, fresh culture medium was replaced and cultured for a further 48 to 96 hours.

[0064] Puromycin was used for resistance screening, and cell clones stably expressing SV40T and HPV genes were selected from the transfected cells and gradually expanded for culture.

[0065] Example 3 Cell proliferation activity assay

[0066] The cell proliferation activity was measured. After 48 hours of culture, the cell status was as shown in the figure Figure 1 As shown, the cell status after 96 h of culture is as follows Figure 2 shown.

[0067] Comparative Example 1

[0068] The immortalized goose ovary granulosa cell line constructed using the SV40 gene was cultured for 48 hours, and the cells under different fields of view were as follows: Figure 3 As shown; the cell status after 96h of culture is shown Figure 4 shown.

[0069] Example 3 Cell state diagram of immortalized goose ovary granulosa cell line constructed using SV40T and HPV genes Compared with the cell state diagram of immortalized goose ovary granulosa cell line constructed using only a single SV40 gene in the comparative example, when the same number of cells were inoculated, the cell line constructed using SV40T and HPV genes proliferated faster, had a higher proliferation efficiency than the immortalized cell line constructed using the SV40 gene, and had a more regular cell morphology. This further shows that the immortalized goose ovary granulosa cell line constructed by the present invention has a higher proliferation efficiency.

[0070] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An immortalized goose ovary granulosa cell line, characterized in that: The cell line is obtained by transfecting a lentiviral vector carrying DNA sequences encoding SV40T and HPV viruses into primary ovarian granulosa cells of a goose; According to the accession number of the DNA sequence encoding the SV40T virus in GenBank, which is NC_001669.1, the primers of the DNA sequence of the SV40T virus are shown in the nucleotide sequence table SEQ ID NO.1-2; the DNA sequence of the SV40T virus is 2075 bp; According to the accession number of the DNA sequence encoding the HPV virus in GenBank, which is NC_001526.4, the primers of the DNA sequence of the HPV virus are shown in the nucleotide sequence table SEQ ID NO.3-4; the DNA sequence of the HPV virus is 729 bp.

2. The goose ovary granulosa cell line according to claim 1, characterized in that: The lentiviral vector is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro.

3. A method for constructing an immortalized goose ovary granulosa cell line, characterized in that: The method comprises using the lentiviral vector of claim 1 to mediate the DNA sequences encoding SV40T and HPV viruses, and transfecting them into host cells, and then screening them with puromycin; the host cells are goose ovary granulosa cells; According to the accession number of the DNA sequence encoding the SV40T virus in GenBank, which is NC_001669.1, the primers of the DNA sequence of the SV40T virus are shown in the nucleotide sequence table SEQ ID NO.1-2; the DNA sequence of the SV40T virus is 2075 bp; According to the accession number of the DNA sequence encoding the HPV virus in GenBank, which is NC_001526.4, the primers of the DNA sequence of the HPV virus are shown in the nucleotide sequence table SEQ ID NO.3-4; the DNA sequence of the HPV virus is 729 bp.

4. The method according to claim 3, characterized in that: The following steps are involved: S1. constructing the DNA sequences encoding SV40T and HPV viruses into the lentiviral vector of claim 1 to obtain a recombinant lentiviral vector; S2, co-transfecting the recombinant lentiviral vector described in S1 with the lentiviral packaging helper plasmid into 293T cells for pseudovirus packaging; S3. The pseudovirus of S2 was transfected into primary isolated goose ovary granulosa cells to obtain an immortalized cell line.

5. The method according to claim 4, characterized in that The lentiviral vector is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro; the lentiviral packaging auxiliary plasmids are pMD2.G and psPAX2.

6. The method according to claim 5, characterized in that The molar ratio of the recombinant lentiviral vector and the lentiviral packaging helper plasmid is pCDH-MSCV-MCS-EF1-CopGFP-T2A-Puro-HPV-SV40T:pMD2.G:psPAX2=4:1:

3.

7. The method according to any one of claims 4 to 6, characterized in that: The method also includes a step of measuring the cell proliferation activity of the immortalized cell line obtained.

8. Use of the immortalized goose ovarian granulosa cell line according to claim 1 or the cell line constructed by the construction method according to claim 3 in goose disease research, wherein the goose is a scimitar goose.

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