CN112522204A - Recombinant MARC-145 cell and construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant MARC-145 cell and a construction method and application thereof, belonging to the technical field of genetic engineering. The invention discloses a recombinant MARC-145 cell, which is MARC-145 for simultaneously expressing porcine CD163 and sialic acid combined Ig-like lectin 10 genes in the MARC-145 cell^{pCD163‑pSiglec10}A cell line. MARC-145 constructed in the invention^{pCD163‑pSiglec10}The cell line can rapidly proliferate PRRSV, and lays a foundation for PRRSV vaccine development and related researchA foundation.

Description

Recombinant MARC-145 cell and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant MARC-145 cell and a construction method and application thereof.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a global viral infectious disease of pigs caused by PRRS Virus (PRRSV), is mainly characterized by sow Reproductive failure and serious Respiratory diseases of piglets, and becomes one of the most serious diseases harming the swine industry in China. Causing great economic loss to the pig industry in China.

PRRSV has a rather strict cell tropism, of which primary Porcine Alveolar Macrophages (PAMs) are the main host cells, in addition to which PRRSV infects mainly monkey kidney cells MA-104 and cell lines derived therefrom in vitro, such as the african green monkey kidney cell line Marc-145 cells. Studies have shown that the cell tropism of PRRSV is largely determined by the receptors for the virus on the cells. Three PRRSV receptors have been identified on PAM cells: heparan Sulfate (HS), sialoadhesin (Siglec-1, CD169), CD163, and the like. On the native host cell PAM, three receptors for PRRSV have been identified: heparan sulfate, Siglec-1 and CD 163. Heparan sulfate is mainly used for adsorbing virus particles, while Siglec-1 can adsorb viruses and mediate endocytosis of the viruses, and the endocytosis is independent of heparan sulfate. In addition, CD163 may assist Siglec-1 endocytosis, viral uncoating and release of genomic RNA into the cytoplasm, and thus the Siglec-1 and CDl63 molecules have a synergistic effect in mediating RRSV entry into PAM. Siglec-10 is a sialic acid binding protein belonging to the same family as Siglec-1, and recent studies show that Siglec-10 is also involved in endocytosis of PRRSV and plays an important role in the process of PRRSV entering cells. The cell line currently used for isolation of PRRSV is the kidney cell line Marc-145 cell of the continuous cell line African green monkey, but Marc-145 expresses only the CD163 receptor and not the Siglec-10 receptor, resulting in that part of PRRSV cannot be isolated on Marc-145 cells at present. Because PAMS which is most easily infected and can isolate PRRSV can only be primarily cultured, the preparation process is troublesome, the cost is high, and the PAMS is easily polluted by other microorganisms, while the currently commonly used MARC-145 cell can proliferate PRRSV, but is derived from monkeys, which is limited by natural hosts, and has the limitations of slow proliferation speed and low titer of PRRSV. Based on this, it is urgent to establish a porcine cell for the isolation culture of PRRSV and related research.

Therefore, it is a problem to be solved by those skilled in the art to provide a recombinant MARC-145 cell and a method for constructing and using the same.

Disclosure of Invention

In view of the above, the invention provides a recombinant MARC-145 cell for simultaneously expressing porcine CD163 and sialic acid binding Ig-like lectin 10(Siglec-10) genes, and a construction method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a recombinant MARC-145 cell is MARC-145 which expresses porcine CD163 and sialic acid binding Ig-like lectin 10 gene simultaneously in MARC-145 cell^{pCD163-pSiglec10}A cell line.

Further, the construction method of the recombinant MARC-145 cell comprises the following specific steps:

(1) PCR is respectively used for amplifying pCD163 and pSiglec-10 genes;

(2) respectively constructing pLVX-pCD163-PGK-Puro and pCDNA3.1-Siglec-10 recombinant plasmids;

(3) and (3) slow virus packaging: introducing the recombinant plasmid pLVX-pCD163-PGK-Puro and the system packaging plasmid into 293T cells together to obtain rLV-pCD 163;

(4) rLV-pCD163 is added into Marc145 cells for incubation, a stable transfectant strain is screened by using complete culture medium containing puromycin, and a polyclonal stable transfectant cell strain MARC-145 is obtained by passage^pCD163；

(5) Recombinant plasmid pCDNA3.1-pSiglec-10 transfection MARC-145^pCD163Cells, screened by G418 to obtain MARC-145^{pCD163-pSiglec10}A cell line.

Further, the sequences of the primers used for PCR amplification of the pCD163 gene in step (1) are as follows:

P1：5’-CTCGAGATGGACAAACTCAGAATGGTG-3’；SEQ ID NO.1；

P2：5’-GGATCCTCATTGTACTTCAGAGTGGTC-3’；SEQ ID NO.2；

the primer sequences for PCR amplification of the pSiglec-10 gene are as follows:

P3：5’-CTCGAGATGCTCCTGCCGCTGCTCTTA-3’；SEQ ID NO.3；

P4：5’-GGTACCTGGAACTGGACCGCAGCATAT-3’；SEQ ID NO.4。

further, the MARC-145^{pCD163-pSiglec10}Application of cell line in improving PRRSV separation rate.

According to the technical scheme, compared with the prior art, the invention discloses and provides the recombinant MARC-145 cell and the construction method and application thereof, the infection capacity of PRRSV has direct correlation with the expression level of a CD163 receptor, and based on the direct correlation, the porcine CD163 and Siglec-10 genes are transfected into the MARC-145 cell to construct the MARC-145 cell capable of efficiently and stably expressing the CD163 and Siglec-10 genes^{pCD163-pSiglec10}The cell line can rapidly proliferate PRRSV, and lays a foundation for PRRSV vaccine development and related research.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing a map of a pLVX-PGK-Puro vector of the present invention;

FIG. 2 is a diagram showing the morphological observation of 293T cells transfected with pLVX-PGK-Puro for 24 h;

FIG. 3 is a diagram showing the morphology observation of 293T cells transfected with pLVX-pCD163-PGK-Puro 48 h;

FIG. 4 is a diagram showing the morphology of rLV-pCD163 virus-infected cells for 48 h;

FIG. 5 is a diagram showing a morphological observation of rLV-PP virus-infected cells for 48h according to the present invention;

FIG. 6 is a diagram showing the morphology of cells after completion of drug screening of Marc145 cells stably transformed by rLV-pCD163 virus according to the present invention;

FIG. 7 is a diagram showing a morphology observation of a cell state when a drug screen for stably transferring Marc145 cells by rLV-PP virus is completed;

FIG. 8 is a diagram showing the result of PCR identification of pCD163 according to the present invention;

m, DL2000 Marker, among others; 1. amplifying the band;

FIG. 9 is a drawing of MARC-145 according to the invention^pCD163Observing the state and morphology of the cells before the mixed clone is frozen;

FIG. 10 is a diagram showing a morphological observation of a cell state before cryopreservation of a mixed clone obtained by stably transforming Marc145 cells with rLV-PP virus according to the present invention;

FIG. 11 is a drawing showing the identification of Marc-145 and Marc145 by Western Blot according to the invention^pCD163Graph of CD163 expression in cells;

wherein, 1, MARC-145; 2. MARC-145^pCD163；

FIG. 12 is a diagram showing the result of PCR identification of Siglec-10 gene according to the present invention;

m, DL2000 Marker, among others; 1. amplifying the band;

FIG. 13 is a drawing showing the identification of Marc-145 and Marc145 by Western Blot according to the invention^{pCD163-pSiglec10}A graph of the expression results of pSiglec10 in cells;

wherein, 1, MARC-145^pCD163；2、MARC-145^{pCD163-pSiglec10}。

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

RNA extraction kits were purchased from Tiangen Biochemical technology Ltd, Superscript III reverse transcriptase and

pfx DNA polymerase was purchased from Invitrogen; porcine Alveolar Macrophages (PAMs) were obtained from piglet alveolar lavage fluid that was negative for both PRRSV, PCV2 at 4 weeks of age.

Example 1 extraction of Total RNA and amplification of pCD163 and pSiglec-10 genes

(1) Extraction of Total RNA

Extracting total RNA of alveolar macrophage (PAMs) cells according to the instruction of an RNA extraction kit, and performing reverse transcription reaction in a system of 20 mu L of SuperScript III reverse transcriptase product instruction, 8 mu L of RNA, 201 mu L of oligo (dT), 1 mu L of dNTP (10mmol/L) and ddH₂O2 μ L, mixing, water bathing at 65 deg.C for 5min, and standing on ice for 2 min; 5 XFirst-Strand Buffer4 μ L, 0.1M DTT 1 μ L, RNaseOUT were added^TMRecombinant RNase Inhibitor 1μL，SuperScript^TMIII RT (200 units/. mu.L) 2. mu.L, mixing, water bathing at 55 deg.C for 1h, inactivating reverse transcriptase in water bathing at 70 deg.C for 15min, and storing the synthesized cDNA at-20 deg.C or using in PCR amplification.

(2) PCR amplification of pCD163 Gene and pSiglec-10 Gene

The reaction system is 50 mu L, PCR amplification reference

The pfx DNA polymerase product was described. To the PCR tube were added 10 Xpfx Amplification Buffer 5. mu.L, 10mM dNTP mix 1.5. mu.L, 50mM MgSO 5. mu.L in this order ₄1 μ L of each of the upstream and downstream primers (10 μ M), 2 μ L of cDNA template, 1 μ L of Pfx DNA Polymerase, ddH₂And O is supplemented to 50 mu L. PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; extension at 94 ℃ for 15s and 68 ℃ for 1min for 30 cycles. After the reaction, the PCR product was identified by electrophoresis on a 1% agarose gel.

Wherein, the primer sequences for amplifying the pCD163 gene are as follows:

P1：5’-CTCGAGATGGACAAACTCAGAATGGTG-3'; SEQ ID No. 1; (Xho I cleavage sites underlined);

P2：5’-GGATCCTCATTGTACTTCAGAGTGGTC-3'; SEQ ID No. 2; (BamHI sites underlined);

the primer sequences for amplification of the pSiglec-10 gene are as follows:

P3：5’-CTCGAGATGCTCCTGCCGCTGCTCTTA-3'; SEQ ID No. 3; (Xho I cleavage sites underlined);

P4：5’-GGTACCTGGAACTGGACCGCAGCATAT-3'; SEQ ID No. 4; (the Kpn I cleavage site is underlined).

Example 2 construction of pLVX-pCD163-PGK-Puro and pCDNA3.1-Siglec-10 recombinant plasmids

PCR product of pCD163 gene and pLVX-PGK-Puro vector (FIG. 1) were digested with Xho I/BamHI, the pCD163 gene and vector gene fragment obtained by recovering the gel were ligated with T4 DNA Ligase, T4 DNA 9. mu.L, 5 XLigase Reaction Buffer 2. mu.L, pCD163 recovered product 6. mu.L, pLVX-PGK-Puro vector plasmid 3. mu.L, ddH₂O8. mu.L, the mixture was ligated at 22 ℃ for 2 hours, and then recombinant plasmid was transformed into E.coli DH-5. alpha. competent cells. Meanwhile, BamHI/Kpn I is used for respectively carrying out double digestion on the PCR product of the Siglec-10 gene and the pCDNA3.1 vector, the gel is recovered to obtain the Siglec-10 gene and a vector gene fragment, the Siglec-10 gene and the vector gene fragment are connected by T4 DNA Ligase, T4 DNA 9 muL, 5 XLigase Reaction Buffer 2 muL, the recovered pSiglec-10 product is 6 muL, the pCDNA3.1 vector plasmid is 3 muL, ddH₂O8. mu.L, the mixture was ligated at 22 ℃ for 2 hours, and then recombinant plasmid was transformed into E.coli DH-5. alpha. competent cells. Selecting a single colony to be inoculated in an LB liquid culture medium, and carrying out PCR and double enzyme digestion identification after shaking culture at 37 ℃. The positive recombinant bacteria are sent to a sequencing company for sequencing.

Example 3 Lentiviral packaging

Introducing a recombinant plasmid pLVX-pCD163-PGK-Puro carrying a target gene, a plasmid pSPAX2 and a plasmid pMD2.G (Huayuyo Biotechnology Co., Ltd.) into 293T cells together to generate a high-titer lentivirus (hereinafter, referred to as rLV-pCD163) carrying the target gene; meanwhile, the recombinant plasmid pLVX-PGK-Puro is used as a control virus (rLV-PP for short). The specific operation is carried out according to the specification of a lentivirus packaging kit (Huayue biology (Beijing) science and technology Co., Ltd.).

The 24h morphological observation chart of 293T cell transfected pLVX-PGK-Puro is shown in FIG. 2, and the 48h morphological observation chart of 293T cell transfected pLVX-pCD163-PGK-Puro is shown in FIG. 3.

EXAMPLE 4 construction of stably transfected pCD163 cell line

(1) Recovering the Marc145 cells, and continuously carrying out passage for 2-3 times when the cells grow to 80% -90%;

(2) culturing Marc145 cells, and counting the cells after the cells grow to 80% -90% of the digested cells according to the ratio of 6x10⁵cells/well cell density seeded cells into 6-well plates;

(3) the next day serum-free medium was changed and virus (rLV-pCD163 or rLV-PP) was added at MOI 10; incubating in an incubator for 2h, and changing to DMEM complete medium (Thermo Fisher Scientific) for culture;

the morphology of the rLV-pCD163 virus-infected cells at 48h is shown in FIG. 4; the 48h morphological observation chart of the rLV-PP virus infected cell is shown in figure 5.

(4) After 48h, DMEM complete medium with puromycin at a concentration of 6. mu.g/ml was added to the selected cells, and every 2 days the medium was changed (DMEM complete medium with puromycin at a concentration of 6. mu.g/ml); meanwhile, blank cells are used as a control group experiment;

(5) when the cells of the control group are completely dead, changing the cells into puromycin DMEM complete culture medium with the concentration of 2 mu g/ml for culture;

the morphology observation picture of the cell state when the drug screening of the rLV-pCD163 virus stable Marc145 cells is completed is shown in FIG. 6; the morphology observation picture of the cell state when the drug screening of the Marc145 cells stably transformed by the rLV-PP virus is finished is shown in figure 7.

(6) After the cells grow full, digesting the cells, carrying out amplification culture, and extracting part of the cells to extract a genome for PCR identification;

the PCR identification primer sequence of the stable transformant is as follows:

CD163-RT3001-F：5’-GAAACCTCCTTGTGGGATTGT-3’；SEQ ID NO.5；

XWF-PGK-R：5’-GCCTACCGGTGGATGTGGAAT-3’；SEQ ID NO.6；

the amplified product was subjected to 1% agarose gel electrophoresis, and the result is shown in FIG. 8, where the amplified fragment size was 510bp, which is identical to the expected target gene size.

(7) Continuously transmitting for 2-3 generations to obtain a polyclonal stably-transformed cell strain MARC-145^pCD163；

MARC-145^pCD163The observation picture of the state of the cells before the mixed clone is frozen is shown in figure 9; the morphology observation picture of the cell state before cryopreservation of the mixed clone obtained by stably transforming Marc145 cells with rLV-PP virus is shown in FIG. 10.

(8) Westernblot detection of CD163 protein expression: MARC-145 screened by lysis with RIPA cell lysate (Biyuntian Biotechnology Ltd.)^pCD163After the cells were cultured, 10% SDS-PAGE was performed, and after completion, the proteins were transferred to an NC membrane, blocked with 5% skimmed milk powder at 37 ℃ for 2 hours, incubated overnight at 4 ℃ with a mouse anti-porcine CD163 monoclonal antibody (abd serotec Co., Ltd.) as a primary antibody (1: 1600 dilution), PBST was washed 3 times, HRP-labeled goat anti-mouse IgG was added as a secondary antibody (1: 3000 dilution) (Bilun sky Biotechnology Co., Ltd.), incubated at 37 ℃ for 2 hours, washed 3 times with PBST, and the results were observed after the color development of a chemiluminescence kit, and the results are shown in FIG. 11.

Example 5 construction of Steady transgenic MARC-145^{pCD163-pSiglec10}Cell line

(1) Recombinant plasmid pCDNA3.1-pSiglec-10 transfection MARC-145^pCD163Cells

MARC-145 was administered the day before transfection experiments^pCD163The cells were spread evenly on a cell culture dish and placed in a cell incubator (37 ℃ C., 5% CO)₂) And (3) culturing overnight, and performing a transfection experiment when the cells grow to about 80% on the next day: firstly, the DMEM complete culture medium in a cell culture dish is replaced by a new DMEM complete culture medium (containing 10% of serum and 1% of double-antibody streptomycin), then a new sterile centrifuge tube is taken, 250 mu L of DMEM complete culture medium without double-antibody and serum is added, 2.5 mu g of endotoxin-free recombinant plasmid pCDNA3.1-Siglec-10 is added, a pipette is used for gently blowing and beating the recombinant plasmid and the DMEM culture solution, finally 8 mu L of Lip8000TM transfection reagent (Biyuntian biotechnology limited) is added, all mixed solution is sucked into the cell culture dish after being gently mixed, and the mixed solution is cultured in a cell culture box for 48 hours. At the same time, set untransfected MARC-145^pCD163And (4) cell control.

(2)MARC-145^{pCD163-pSiglec10}Selection of cell lines

After 24h of transfection, the screened 600 mug/mL G418 is added into the DMEM complete medium, the fresh medium is changed every 3d, the selection is carried out until the 6 th day, the control group dies completely, the survived monoclonal antibody cells are picked and expanded for culture, the cells are diluted by a limiting dilution method, a 96-well cell culture plate is inoculated, the number of the cells in each well is 1-3, and the DMEM complete medium containing the G418(600 mug/mL) is changed every 3d 1 time. The obtained monoclonal cell line was expanded and cultured after 3 cell subcloning. After the cells are full, digesting the cells, carrying out amplification culture, taking partial cells, extracting a genome, and carrying out PCR identification by using a P3/P4 primer, wherein the size of a band is 1800 bp. The results are shown in FIG. 12.

(3) Westernblot for detecting pSiglec10 protein expression

MARC-145 screened by lysis with RIPA cell lysate (Biyuntian Biotechnology Ltd.)^{pCD163-pSiglec10}After the cells were cultured, 10% SDS-PAGE was performed, and after the completion, the proteins were transferred to an NC membrane, blocked with 5% skim milk powder at 37 ℃ for 2 hours, incubated overnight at 4 ℃ with pSiglec10 polyclonal antibody (1: 800 dilution) (prepared in the livestock infectious disease control and biotechnology focus laboratory of Fujian province) as the primary antibody, PBST was washed 3 times, HRP-labeled goat anti-mouse IgG was added as the secondary antibody (1: 3000 dilution) (Biyuntian Biotechnology Co., Ltd.), incubated at 37 ℃ for 2 hours, washed 3 times with PBST, and the results were observed after the color development with a chemiluminescence kit, and the results are shown in FIG. 13.

Example 6MARC-145^{pCD163-pSiglec10}Application of cell line

100 parts of PRRSV pathogenic material samples with positive RT-PCR detection in the clinic of 2016-2018 in the laboratory are inoculated with MARC-145 at the same time^{pCD163-pSiglec10}Cells were virus isolated from MARC-145 cells. The results show that MARC-145^{pCD163-pSiglec10}The isolation rates of cells and MARC-145 cells from the virus were 90% and 82%, respectively, as seen in MARC-145^{pCD163-pSiglec10}The cell obviously improves the separation rate of PRRSV, and is more suitable for the separation of clinical PRRSV.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims (4)

1. A recombinant MARC-145 cell expressing both porcine CD163 and sialic acid binding to MARC-145 of Ig-like lectin 10 gene simultaneously in MARC-145 cells^{pCD163-pSiglec10}A cell line.

2. The method of claim 1, wherein the steps of constructing the recombinant MARC-145 cell are as follows:

(1) PCR is respectively used for amplifying pCD163 and pSiglec-10 genes;

(2) respectively constructing pLVX-pCD163-PGK-Puro and pCDNA3.1-Siglec-10 recombinant plasmids;

(3) and (3) slow virus packaging: introducing the recombinant plasmid pLVX-pCD163-PGK-Puro and the system packaging plasmid into 293T cells together to obtain rLV-pCD 163;

(4) rLV-pCD163 is added into Marc145 cells for incubation, a stable transfectant strain is screened by using complete culture medium containing puromycin, and a polyclonal stable transfectant cell strain MARC-145 is obtained by passage^pCD163；

(5) Recombinant plasmid pCDNA3.1-pSiglec-10 transfection MARC-145^pCD163Cells, screened by G418 to obtain MARC-145^{pCD163-pSiglec10}A cell line.

3. The method of claim 2, wherein the primer sequences for PCR amplification of pCD163 gene in step (1) are as follows:

P1：5’-CTCGAGATGGACAAACTCAGAATGGTG-3’；SEQ ID NO.1；

P2：5’-GGATCCTCATTGTACTTCAGAGTGGTC-3’；SEQ ID NO.2；

the primer sequences for PCR amplification of the pSiglec-10 gene are as follows:

P3：5’-CTCGAGATGCTCCTGCCGCTGCTCTTA-3’；SEQ ID NO.3；

P4：5’-GGTACCTGGAACTGGACCGCAGCATAT-3’；SEQ ID NO.4。

4. the MARC-145 of any of claims 1 to 3^{pCD163-pSiglec10}Application of cell line in improving PRRSV separation rate.

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