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Immortalized pig bone marrow macrophage as well as construction method and application thereof

A macrophage and construction method technology, applied in biochemical equipment and methods, microorganism-based methods, applications, etc., can solve the problems of strict growth conditions, difficult passage, limited survival time and yield, etc., and achieve a clear cell outline. , the effect of cell integrity

Pending Publication Date: 2022-04-08
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, life science research often involves two types of cells, one is primary cells, and the other is immortalized cells or cell lines. At present, porcine bone marrow macrophages have strict growth conditions, limited survival time and limited yield when cultured in vitro. , Difficulty in passage and other issues, so it is necessary to use immortalization technology to establish immortalized porcine bone marrow macrophage cell lines, but so far there are few related reports

Method used

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  • Immortalized pig bone marrow macrophage as well as construction method and application thereof
  • Immortalized pig bone marrow macrophage as well as construction method and application thereof
  • Immortalized pig bone marrow macrophage as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Isolation and cultivation of porcine bone marrow macrophages

[0053] Select the femur and tibia of 15-day-old healthy piglets, remove the surface tissue, and be careful not to cut the periosteum during operation to avoid contamination during later washing. The treated bone was soaked in 75% alcohol for 5 minutes, then transferred to an ultra-clean bench and washed with sterile PBS, the two ends of the bone were amputated with the help of bone forceps, and a puncture needle was inserted into both ends for washing. Use a 20mL needle to absorb the 1640 medium containing 10wt% serum and 1wt% antibiotics to wash the bone marrow, and wash each end of the bone once into a 24-well-plate (24-well plate). Centrifuge for 6 minutes and discard the supernatant. Then wash twice with 1640 medium containing 10wt% serum and 1wt% antibiotics, centrifuge at 550×g for 6 minutes at 4°C, discard the supernatant, and then lyse with 5mL erythrocyte lysate for 5 minutes, during whic...

Embodiment 2

[0054] Example 2 Construction of pIED-Neo-SV40TAg plasmid

[0055] In order to construct pIED-Neo-SV40TAg, the SV40TAg gene containing BglII and BCLI sites was synthesized in Nanjing GenScript Biotechnology Co., Ltd., and cloned into the pIED-Neo vector to obtain the pIED-Neo-SV40TAg plasmid (see figure 1 ). After digesting the plasmid, perform gel electrophoresis to verify the size of the target gene, such as figure 2 As shown, the target bands appeared at the positions of 8668bp and 333bp, and the pIED-Neo-SV40TAg plasmid was successfully constructed by sequencing.

Embodiment 3

[0056] Example 3 Construction of pIED-Neo-CD163-CD169-CD151 plasmid

[0057] The codon-optimized CD163-CD169-CD151 gene (SEQ ID NO: 2) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pIED-Neo-SV40TAg vector to obtain pIED-Neo-CD163-CD169-CD151 Plasmid (see image 3 ).

[0058] Specifically, the pIED-Neo-SV40TAg vector is used as a template, and CD163-CD169-CD151-F (shown in SEQ ID NO: 4), CD163-CD169-CD151-R (shown in SEQ ID NO: 5) are used as upstream and downstream The primers were used for PCR amplification, and the amplification system is shown in Table 1.

[0059] Table 1 CD163-CD169-CD151 gene amplification system

[0060]

[0061] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0062] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as...

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Abstract

The invention discloses an immortalized pig bone marrow macrophage as well as a construction method and application thereof. The construction method comprises the following steps: transfecting porcine bone marrow macrophages by using a first recombinant plasmid containing a nucleotide sequence as shown in SEQ ID NO.1, then culturing the transfected cells, and separating out cell monoclone. Furthermore, the immortalized porcine bone marrow macrophage is modified in a manner of transfecting the immortalized porcine bone marrow macrophage with a second recombinant plasmid and a third recombinant plasmid containing nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3. The invention further provides a preparation method of the immortalized porcine bone marrow macrophage. The construction and transformation methods of the immortalized pig bone marrow macrophage line provided by the invention are simple and easy to operate, the obtained immortalized pig bone marrow macrophage line has the remarkable characteristic of continuous passage, and the virus sensitivity is high after the immortalized pig bone marrow macrophage line is transformed.

Description

technical field [0001] The application specifically relates to an immortalized porcine bone marrow macrophage, its construction method and application, and belongs to the field of biotechnology. Background technique [0002] Macrophages are one of the important immune defense cells of the body. They are widely distributed throughout the body and exist in lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bones and skin. They are important for host innate and specific immunity. It is also a key regulator and effector cell in the immune response (Verdeguer F and Aouadi M. Macrophageheterogeneity and energy metabolism [J]. ExpCellRes, 2017, 360(1): 35-40). In non-specific immunity, macrophages can kill and remove pathogens and foreign substances through phagocytosis, mediate inflammatory responses, and participate in innate immune responses; and, in specific immunity, process and present antigens as antigen-presenting cells and Initiate an adaptive ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/37C12N15/12C12R1/91
Inventor 张大鹤滕小锘王俊肖徐龙飞
Owner 苏州沃美生物有限公司
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