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
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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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