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CD86-based membrane fixed protein, and preparation method and application thereof

A technology of CD86 and membrane immobilization, which is applied in the field of genetic engineering, can solve the problems of limited amplification, unsuitable for large-scale amplification, expensive human serum, etc., and achieve the effect of high expression

Inactive Publication Date: 2017-03-15
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current problem in immune cell therapy is to obtain a certain immune cell subgroup with a certain purity and quantity. Usually, various cytokine combinations and magnetic bead sorting methods are used. This method requires a large amount of peripheral blood collected from the patient and limited amplification
The existing media on the market are not formulated for the conditions required for immune cell culture, resulting in poor cell proliferation efficiency and biological efficacy, and many media are supplemented with human or animal serum and components to fully supplement
However, human serum is expensive and not suitable for large-scale amplification, and due to the heterogeneity of donors, there are differences in the quality of serum between batches, especially the risk of contamination by hepatitis virus or HIV
The serum-free medium added with human serum albumin, human transferrin, human insulin and other human-derived components, although the source of the disease has been detected and sterilized, it is still unavoidable that there are differences between batches due to different donors, which is not conducive to quality control
Animal serum such as fetal bovine serum, newborn bovine serum and other serum components from bovine materials, although there is sufficient supply and low price, there is a risk of spreading infectious diseases, such as mycoplasma, virus (such as the risk of BSE pathogenic factor) contamination risk

Method used

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  • CD86-based membrane fixed protein, and preparation method and application thereof
  • CD86-based membrane fixed protein, and preparation method and application thereof
  • CD86-based membrane fixed protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of lentiviral expression vector pCDH-SP86CD64

[0051] The SP86CD64 target gene was synthesized by Jiman Biotechnology Co., Ltd., the sequence is shown in SEQ ID NO.1, and it was connected to the pUC57 vector;

[0052] (1) Design primers according to the known sequence of pUC57-SP86CD64 as follows:

[0053] Upstream primer: 5'-GCGGATCCATGGACCCCCAGT-3' (SEQ ID NO.2);

[0054] Downstream primer: 5'-ATAAGAATGCGGCCGCTTAAAAACATGT-3' (SEQ ID NO.3);

[0055] The pUC57-SP86CD64 vector was used as a template for PCR amplification, and the PCR amplification reaction system was as follows:

[0056]

[0057] Wherein, water is used as a sample as a negative control.

[0058] The reaction conditions are as follows:

[0059]

[0060] The obtained PCR product is identified by 1% agarose gel electrophoresis, and the obtained electrophoresis detection result is as follows: figure 1 As shown, it can be seen that SP86CD64 is 1170bp, which has been connect...

Embodiment 2

[0068] Example 2: Plasmid extraction

[0069] The existing control vector LV5-EGFP, the target gene expression vector pCDH-SP86CD64, and the packaging plasmid engineering bacteria were cultured, and then the plasmids to be extracted were: the control vector LV5-EGFP, the target gene expression vector pCDH- SP86CD64, packaging plasmids PSPAX2 and PMD2G.

[0070] Dali uses the SDS alkaline lysis method, combined with silica gel mold filter membrane, to obtain a high-purity and endotoxin-free plasmid DNA solution from the overnight culture of LB medium. The plasmid is dissolved in Tris Buffer, and the obtained plasmid can be used directly. For transformation, sequencing, transcription, and enzyme digestion reactions. The specific steps are: take out the corresponding strains from -80°C, streak on the plate, and culture overnight for 12-24 hours; pick 2-4 single clones the next day, and shake the bacteria in a small volume of 5mL for 6-8 hours; Take the bacterial solution and sh...

Embodiment 3

[0071] Example 3: Calcium carbonate virus packaging

[0072] Co-transfect 293T cells with the constructed lentiviral expression vector and packaging plasmid, package the virus, collect and concentrate the virus liquid, store and dilute, and perform titer detection and MOI value determination.

[0073] This experimental method uses the calcium phosphate transfection method for transfection. Viruses were collected and concentrated by centrifugal ultrafiltration. The determination of the virus titer adopts the unsaturated infection flow method, and the specific steps are as follows:

[0074] Inoculate 293T cells and count wells of a six-well plate to make the degree of fusion reach 70% after attachment, and count the inoculated cells as N; 6-8 hours after cell attachment, add virus solution V (such as 10, 7.5, 5, 2.5 μ l), the infection percentage of each gradient is detected by flow cytometry, and the infection group close to 50% is selected, and the infection rate is M; accor...

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Abstract

The invention relates to the technical field of gene engineering, in particular to CD86-based membrane fixed protein, and a preparation method and application thereof. CD86 comprises a signal peptide encoding gene, an extracellular region encoding gene, a transmembrane region encoding gene and an intracellular region encoding gene; and the membrane fixed protein is to replace the extracellular region encoding gene of the CD86 with an expressible exogenous gene. Lentivirus-containing host cells can be used for cultivating cells, and can supply nutritional substances for the cells continuously, additional nutritional substances are not needed, and convenience and rapidness are realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a membrane-fixed protein based on CD86, its preparation method and application. Background technique [0002] Adoptive cellular immunotherapy (ACI or AIT) of tumors refers to the transfer of immune cells (specific and relatively specific) with anti-tumor activity to tumor patients to directly kill tumors or stimulate the body's immune response to kill tumor cells , to achieve the purpose of treating tumors. The cell immunotherapy is an emerging and brand-new anti-tumor treatment method with significant curative effect in tumor rehabilitation medicine. It is known as the most active and promising treatment method in the comprehensive tumor treatment model in the 21st century. [0003] The current problem in immune cell therapy is to obtain a certain immune cell subgroup with a certain purity and quantity. Usually, various cytokine combinations and magnetic bead sortin...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N7/01C12N5/10C12N15/867C12N5/0783C12R1/93
CPCC07K14/70532C07K14/54C07K14/5443C07K14/70503C07K14/70535C07K14/70539C07K14/70578C07K2319/02C07K2319/03C07K2319/32C12N5/0636C12N5/0637C12N5/0646C12N7/00C12N15/86C12N2501/2315C12N2501/2321C12N2501/51C12N2501/599C12N2502/99C12N2740/15021C12N2740/15043
Inventor 韦丹秦志华连杰马亚锋范宇清郭峰
Owner HENAN HUALONG BIOLOGICAL TECH
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