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Recombinant plasmid stably expressing CD163 receptor protein, recombinant lentivirus and porcine alveolar macrophage cell line and construction method thereof

A technology of alveolar macrophages and recombinant lentivirus, applied in the direction of receptors/cell surface antigens/cell surface determinants, viruses/bacteriophages, chemical instruments and methods, etc., can solve the problem of pathogenic microorganisms invading the body, difficult to obtain pPAM, Virus persistent infection and other problems, to achieve the effect of improving industrial production efficiency

Inactive Publication Date: 2019-09-03
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the virus is very prone to mutation, prone to immune evasion, persistent virus infection, and easy to induce a wide range of secondary infections, allowing other pathogenic microorganisms to invade the body
[0004] However, since pPAM cells are not only difficult to obtain, but also cannot be conveniently preserved or used for long-term
Meanwhile, in some PRRSV endemic countries (such as China), it is difficult to obtain pPAM derived from PRRS antigen and antibody double-negative pigs
Although an immortalized PAM cell line (mPAM) has been reported, this cell line cannot be infected by PRRSV because it cannot express the receptor protein CD163 required for PRRSV infection
Therefore, this severely restricts the research on the mechanism of interaction between infected PAM and host immune cells, and it is difficult to effectively control PRRSV
On the other hand, the cells currently used in the industrialized production of PRRSV vaccines are MARC-145 cells, and the toxicity of highly pathogenic PRRSV (HP-PRRSV) on the cells is relatively low, generally 10 4 -10 6 TCID 50 / 0.1mL, leading to a large room for improvement in the production efficiency of the vaccine

Method used

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  • Recombinant plasmid stably expressing CD163 receptor protein, recombinant lentivirus and porcine alveolar macrophage cell line and construction method thereof
  • Recombinant plasmid stably expressing CD163 receptor protein, recombinant lentivirus and porcine alveolar macrophage cell line and construction method thereof
  • Recombinant plasmid stably expressing CD163 receptor protein, recombinant lentivirus and porcine alveolar macrophage cell line and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] This embodiment provides a recombinant plasmid (named pLV-EGFP-CD163) for the expression of CD163 receptor, and its construction method is as follows:

[0053] 1. Cloning and sequencing of CD163 gene:

[0054] Obtain pPAM cells from the PRRSV negative piglet lung of 4-6 weeks of age, design primer (primer sequence as shown in table 1) according to the gene sequence of pig CD163, utilize PCR method to amplify pig CD163 gene (the reaction system of PCR amplification and The reaction conditions are shown in Table 2 and Table 3).

[0055] Table 1 is used to amplify the primer of CD163 gene

[0056]

[0057] Table 2 PCR reaction system

[0058]

[0059]

[0060] Table 3 PCR reaction conditions

[0061]

[0062] 2. Construct the cloning plasmid pEASY-T1-CD163:

[0063] The porcine CD163 gene was ligated with the cloning vector pEASY-T1 (the ligation system is shown in Table 4), and the obtained cloning plasmid was sequenced, and the sequencing results were com...

Embodiment 2

[0078] This example provides a recombinant lentivirus and porcine alveolar macrophage cell line stably expressing CD163 receptor expression, the construction method of which is as follows:

[0079] 1. Preparation of recombinant lentivirus:

[0080] Lenti-X 293T cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO2. 24 hours before transfection, logarithmic phase 293T cells were digested with trypsin, inoculated in 100 mm tissue culture dishes (medium volume 8 mL), shaken gently, and incubated at 37° C. with 5% CO 2 . Transfection experiments were performed when the cell density reached 80%-90%.

[0081] Dilute lentiviral vector plasmid (pLV-EGFP-CD163) DNA with sterile water and mix well by vortexing. The resulting DNA solution was added to Lenti-X packaging single injection (VSV-G) tubes. Cap tightly and vortex the resulting solution for 20 s. After incubating at room temperature for 10 min, the DNA solution was slowly added dropwise to the 293T cell solution,...

experiment example

[0102] The identification and performance evaluation of the porcine alveolar macrophage cell line mPAM-CD163 stably expressing the CD163 receptor provided in Example 2 of the present invention are as follows:

[0103] 1. Western blot analysis:

[0104] Monolayers of cells washed with PBS were lysed with RIPA (Beyotime) lyase supplemented with protease inhibitors to prepare whole cell lysates. After boiling for 5 minutes, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein bands were transferred to nitrocellulose filters. After blocking with 5% skim milk for 1 hour at room temperature, the membrane was incubated overnight at 4°C with the primary antibody rabbit anti-CD163 polyclonal antibody (1:1000 dilution). Then, the membrane was incubated with goat anti-rabbit secondary antibody (Earthox) (1:5000 dilution) for 1 hour at room temperature. Finally, a color solution is added to protect from light.

[0105] Test results su...

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Abstract

The invention provides a recombinant plasmid stably expressing CD163 receptor protein, recombinant lentivirus and a porcine alveolar macrophage cell line and a construction method thereof, which belong to the technical field of biology. The recombinant plasmid is constructed based on a lentiviral expression vector pLV-sfGFP[2A]Puro and integrated with a CD163-encoding gene. According to the recombinant plasmid stably expressing the CD163 receptor protein, the recombinant lentivirus and the porcine alveolar macrophage cell line and the method for constructing the same. The CD163 gene is integrated into an immortalized PAM cell chromosome using a special lentiviral system, so that the immortalized PAM cell line can permanently and stably express CD163 protein, and the cells are susceptible to PRRSV infection. The virulence of PRRSV on the cell line (mPAM-CD163) can be as high as 106.9 TCID50 / 0.1 mL, and realizes stable passage.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant plasmid stably expressing CD163 receptor protein, a recombinant lentivirus, a porcine alveolar macrophage cell line and a construction method thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) emerged in Europe and the United States in the early 1990s and has since become a serious problem in the global swine industry. PRRS is a persistent, severe disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) that is characterized by respiratory problems in pigs of different ages, poor growth performance, and reproductive failure in pregnant sows. Measures to control the disease are complicated by the persistent subclinical infection of the disease and the high variability of the virus and antibody responses that are not completely immune to viral reinfection and reemergence. At present, PRRSV exists in almost eve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N5/10C12N15/12
CPCC07K14/705C12N15/86C12N2740/15043
Inventor 彭军徐煜琳庞恒李传刚王亭亭吴家强
Owner SHANDONG AGRICULTURAL UNIVERSITY
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