Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof
A NKT cell, multi-stimulatory technology, applied in recombinant DNA technology, animal cells, vertebrate cells, etc., can solve the problems of high cost, poor repeatability, complex preparation process, etc., and achieve the effect of low cost, high efficiency and easy preparation
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[0052] The present invention will be described in detail below in conjunction with the examples, but they are not further limiting the present invention.
[0053] 1. Preparation of ME64 cells: Obtain CD64 cDNA, perform PCR amplification on CD64 cDNA, obtain the PCR amplification product of CD64 cDNA, then construct prokaryotic expression vector pGEMT-CD64, and further construct its eukaryotic expression vector pcDNA3.1(+) -CD64, the recombinant plasmid pcDNA3.1(+)-CD64 was transfected into MEG-01 cells, G418 screening, and FACS sorting to obtain ME64 cells; specifically:
[0054] (1) Acquisition of CD64 cDNA and amplification of target fragments: Take U937 cells in logarithmic phase, routinely extract RNA with Trizol, use Oligod(T)n as a primer, reverse transcribe mRNA to generate cDNA; and then amplify cDNA by PCR , to obtain the PCR amplification product of CD64cDNA, the fragment size is about 1000pb, roughly consistent with the experimental design of 949 bp; as figure 1 As...
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