T cell capable of improving immune response, and preparation method and application thereof
A technology of immune response and immune cells, applied in the field of T cells, can solve the problems of high cost of treatment, chronic viral infection and bacterial infection without treatment methods, etc., and achieve the effect of rapid self-proliferation and high expression
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Embodiment 1
[0039] Embodiment 1, preparation carries the retrovirus expression plasmid of exogenous gene
[0040] 1. Construction of clones: (1) Total RNA was extracted from mouse splenocytes and DC cells (dendritic cells), and used as a cDNA library after reverse transcription. (2) Use the cDNA library as a template to amplify CD40L and 4-1BBL sequences by PCR, use Migr1 as a carrier to amplify the IERS sequence by PCR, and use the 2A sequence synthesized by the company as a template to amplify the 2A sequence by PCR. In this step:
[0041] Primers used in PCR amplification include:
[0042]
[0043] The PCR amplification system is:
[0044]
[0045]
[0046] The procedure used for PCR amplification is:
[0047]
[0048] CD40L sequence (SEQ ID No.19), 4-1BBL sequence (SEQ ID No.20) (see figure 1 ) are as follows:
[0049] SEQ ID No. 19:
[0050] ATGATAGAAACATACAGCCAACCTTCCCCCAGATCCGTGGCAACTGGACTTCCAGCGAGCATGAAGATTTTTATGTATTTACTTACTGTTTTCCTTATCACCCAAATGATTGGATCTGTGCTTT...
Embodiment 2
[0076] Embodiment 2, in OT-I CD8 + T cell surface expresses CD40L and 4-1BBL
[0077] a. 293T is passaged and plated, and the retrovirus is packaged, as in Example 1.
[0078] b. Spleen cells were isolated from 6- to 8-week-old OT-I mice and activated by adding N4.
[0079] c. 24h later, the activated OT-I CD8 was transduced with different retroviruses carrying foreign genes + T cells:
[0080] Treatment ①: The activated OT-I CD8 was transduced with a retrovirus carrying the exogenous gene CD40L alone + T cells;
[0081] Treatment ②: transduction of activated OT-I CD8 with retrovirus carrying foreign gene 4-1BBL alone + T cells;
[0082] Treatment ③ transduction of activated OT-I CD8 with retrovirus carrying exogenous gene 4-1BBL-2A-CD40L + T cells;
[0083] Treatment ④: transduction of activated OT-I CD8 with retrovirus carrying exogenous gene 4-1BBL-IRES-CD40L + T cells;
[0084] Treatment ⑤: transduction of activated OT-I CD8 with retrovirus carrying exogenous g...
Embodiment 3
[0093] Embodiment 3, in OT-I CD8 + Detection of cell proliferation after expressing CD40L and 4-1BBL on the surface of T cells
[0094] a. Take OT-I mice aged 6-8 weeks, separate spleen cells and add N4 to stimulate culture.
[0095] b. OT-I CD8 that will be activated after 24h + T cells are evenly divided, and the CD8 cells activated are respectively transduced with different viruses obtained in Example 1. + T cells.
[0096] c. After another 24 hours, CD8 + T cells are collected and counted under a microscope. Operation process see Figure 11 a.
[0097] For test results, see Figure 11 b. Overexpression of CD40L can promote cell proliferation compared with the control, but there is no statistical difference. Overexpression of 4-1BBL can effectively promote cell proliferation, and the auxiliary effect is stronger when overexpressing 4-1BBL and CD40L at the same time, indicating that 4-1BBL and CD40L have a mutual superimposed effect in promoting cell proliferation...
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