Trophoblast, preparation method thereof and application of trophoblast in massive rapid amplification of gamma delta T cells

A technology of nourishing cells and cells, which is applied in the fields of genetic engineering and cell biology, can solve the problems of low content and low immunogenicity of γδT cells, and achieve the effect of reducing costs

Active Publication Date: 2020-05-29
中邦干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the very low content of Vγ9Vδ2 T cells in umbilical cord blood, coupled with the low immunogenicity of γδ T cells derived f

Method used

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  • Trophoblast, preparation method thereof and application of trophoblast in massive rapid amplification of gamma delta T cells
  • Trophoblast, preparation method thereof and application of trophoblast in massive rapid amplification of gamma delta T cells
  • Trophoblast, preparation method thereof and application of trophoblast in massive rapid amplification of gamma delta T cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Construction of pLenti-okt3-CD64-CD86-4.1BBL lentiviral plasmid vector

[0075] Query on websites such as NCBI to obtain okt3 (as shown in SEQ IDNO:1), CD64 (as shown in SEQ IDNO:2), CD86 (as shown in SEQ IDNO:3), 4.1BBL (as shown in SEQ IDNO:4) The whole gene coding sequence;

[0076] The okt3-E2A-CD64-P2A-CD86-F2A-4.1BBL-T2A-Puro gene was synthesized by gene synthesis, and two restriction sites XbaI and SalI were synthesized on both sides of it, and the coding region avoided these two Restriction sites;

[0077] The above synthesized gene was double digested with XbaI and SalI enzymes, and then connected to the lentiviral vector pLenti-mbIL21-4.1BBL (gifted by the Institute of Genetics and Development, Chinese Academy of Sciences) by T4 DNA ligase to obtain pLenti-okt3-CD64- CD86-4.1BBL (as shown in SEQ ID NO: 5), such as figure 1 shown.

[0078] The ligation product was transformed into E.coli (DH5α) cells, and the positive clones were identified by PCR, and then...

Embodiment 2

[0081] Packaging preparation of recombinant lentivirus

[0082] 1. Add 4.5million 293FT cells and 9ml DMEM complete medium to each 10cm cell culture dish, mix well, and culture in a cell culture incubator (37°C, 5% (v / v) CO 2 );

[0083] 2. On the second day of culture, add the following reagents to each culture dish: 500 μL jetPRIME buffer, 6 μg pLenti-okt3-CD64-CD86-4.1BBL or pLenti-CD64-CD86-4.1BBL plasmid vector, 3 μg psPAX2 and 1.5 μg pMD2.G , mix evenly, then add jetPRIME, 25μL / 10cm petri dish, mix evenly again, and let stand at room temperature for 10min to get the mixed solution;

[0084] 3. Take out the 293FT cells used for packaging the virus from the incubator, add the mixture evenly to each culture dish, mix well, and put it into the incubator to continue culturing (37°C, 5% (v / v) CO 2 ). After culturing for 4 h, discard the old medium, add PBS to wash the cells, then add DMEM complete medium containing 10wt% FBS, and put them in an incubator for cultivation (37...

Embodiment 3

[0088] Preparation of K562-okt3-CD64-CD86-4.1BBL trophoblast cells

[0089] Put 1 million vigorously growing K562 cells into one well of a 24-well plate for culture, and the medium is 1 ml of 1640 containing 10% FBS;

[0090] Then add 300 μL of lentiviral particles concentrated by ultracentrifugation of the above-mentioned 30 ml of virus supernatant into the wells, and culture (37° C., 5% (v / v) CO 2 ) After 5 days, selective culture was carried out with 3 μg / ml of Puromycin;

[0091]Detect the expression of these four genes okt3, D64, D86 and 4.1BBL in K562 cells by flow cytometry, so that the expression of each gene is above 80% (see image 3 );

[0092] Then 100Gy of γ-rays were used to irradiate for 10 minutes, and after irradiation, branches were frozen in a -80°C refrigerator for future γδT cell culture experiments.

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Abstract

The invention relates to a trophoblast, a preparation method thereof and application of the trophoblast in massive rapid amplification of gamma delta T cells. The preparation method comprises the following steps: S1, constructing a pLenti-okt3-CD64-CD86-4.1BBL lentiviral plasmid vector; S2, packaging lentiviral particles by using the pLenti-okt3-CD64-CD86-4.1BBL plasmid vector; S3, preparing K562-okt3-CD64-CD86-4.1BBL trophoblast; S4, obtaining buffy coat cells of umbilical cord blood; and S5, carrying out in vitro amplification on gamma delta T cells of the umbilical cord blood. According tothe preparation method, an okt3 single-chain antibody is expressed on a trophoblast membrane through a CD8A transmembrane region, especially, the gamma delta T cells can be rapidly and massively amplified from the umbilical cord blood at low cost, the amplification multiple of the gamma delta T cells can reach about 10000 times after a 24-day amplification period, and the purity of the prepared gamma delta T cells can reach 90% or above. Compared with stimulation by a soluble okt3 antibody, the membrane-bound okt3 has the amplification multiple over 2 times to the gamma delta T cells, and hasa good clinical application prospect.

Description

technical field [0001] The invention relates to the fields of genetic engineering and cell biology, in particular to a trophoblast, its preparation method and its application in a large number of rapidly expanding γδT cells. Background technique [0002] According to the T cell receptor, T cells can be divided into two types, namely αβ T cells and γδ T cells. In healthy adults, γδT cells account for about 3% to 5% of T cells in peripheral blood, and 50% to 75% of them are Vγ9Vδ2T cells. In cord blood, the content of γδT cells is even lower, possibly only Therefore, it is difficult to expand cord blood γδT cells in large quantities in vitro. What γδT cells mediate is a kind of innate immunity, which does not require the antigen presentation of MHC molecules, and plays an important role in immune surveillance and prevention of tumor occurrence. It has been found to infiltrate various human cancer tissues such as renal, ovarian, and rectal cancers, recognize stress-related an...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N5/0783
CPCC12N15/86C12N5/0694C12N5/0646C12N2740/15043C12N2800/107C12N2502/99
Inventor 张云龙
Owner 中邦干细胞科技有限公司
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