A kind of car-t transgene carrier for alleviating crs that closes IL6R and its construction method and application
A transgenic vector and carrier technology, applied in the field of medical biology, can solve the problems of high cost and unbearable for ordinary families, and achieve the effects of saving expensive costs, avoiding low in vivo delivery efficiency and saving costs.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0065] Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention. Example 1 Construction of recombinant lentiviral vector
[0066] 1. Materials
[0067] 1. The lentiviral backbone plasmid pLenti-3G Basic2, the lentiviral packaging plasmids pPac-GP, pPac-R and the membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzymes were purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;
[0068] 2. Primers: According to the principles of primer design, the primers required for amplifying DNA fragments and target sites are designed. The primers are synthesized by Shanghai Biological Company, specifically:
[0069] EF1α-F: 5'-ATTCAAAATT...
Embodiment 2
[0184] Example 2 Concentration and detection of recombinant lentiviral vector
[0185] 1. Purification of recombinant lentiviral vectors by ion exchange chromatography (such as Figure 7 shown):
[0186] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;
[0187] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;
[0188] (3) Place two ion exchange columns in series, and pass through the columns sequentially with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl25mM Tris-HCl (pH 6-8);
[0189] (4) The solution obtained in step 2 is loaded on the ion exchange column with a speed of 1-10ml / min by a peristaltic pump;
[0190] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;
[0191] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;
[01...
Embodiment 3
[0244] Example 3 Functional detection of recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, and lvCAR19-scFv0.
[0245] 1. Cell-level expression detection of CAR gene:
[0246] (1) After the recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 and the control virus Mock infected PBMC cells, the collected cells were detected by RT-PCR for the mRNA transcription levels of CAR gene and scFv gene, and verified The expression of CAR gene and scFv gene, if the transcription level of CAR gene and scFv gene mRNA increases, it means that the transcription level of CAR gene and scFv gene is successfully expressed;
[0247] (2) After the recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 and the control virus Mock infected PBMC cells, the collected cells were detected by western blot to detect the expression level of CAR protein to verify the expression...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More