Lentivirus recombinant expression vector/recombinant lentivirus, as well as application, host cell and preparing method thereof

[0068] A method for constructing a human-derived estrogen-related receptor alpha overexpression lentiviral recombinant expression vector (pWPXLd-hERRα), comprising the following steps:

[0069] 1. Amplification and purification of hERRα target gene

[0070] 1.1 Primer design and synthesis: use primerpremier5.0 software to design primers as follows:

[0071] F (SEQ ID NO: 2): 5'-GGGTTTAAACATGTCCAGCCAGGTG-3';

[0072] R (SEQ ID NO: 3): 5'-CCCCCGGGTCAGTCCATCATGGCC-3'.

[0073] F and R introduce restriction endonucleases PmeI and SmaI cutting sites, respectively.

[0074] 1.2 Use the pCMX-hERRα plasmid as a template to amplify the target fragment (please modify the yellow highlighted template) Use the 500-fold diluted pCMX-hERRα plasmid as a template, and use the above primers for PCR amplification. The reaction conditions for amplification are: 94°C Pre-denatured for 2 minutes, denatured at 98°C for 10 seconds, annealed at 59°C for 30 seconds, extended at 68°C for 1 minute and...

[0164] A method for expressing an estrogen-related receptor alpha overexpression lentiviral recombinant expression vector (pWPXLd-hERRα) in 293T and HepG2 cell lines, comprising the following steps:

[0165] 3.1 Lentivirus packaging, concentration and infection

[0166] 1) 293FT cells were plated one day in advance (use DMEM complete medium with ordinary pH 7.4 when plated), and the 293FT cells normally cultured in a 6cm plate with a density of about 85-95% were divided into two new 6cm plates ( About 6*105 cells), or directly plate all in a 10cm plate; 12-18h after plating is the best transfection time, if it exceeds 24h, do not transfect, it is recommended to re-plate;

[0167] 2) Transfection: Before transfection, replace the liquid with DMEM complete medium at pH 7.4 (add 3ml of medium to a 6cm plate)

[0168] Take two 1.5ml Dorf tubes, add 300ul DMEM complete medium respectively, dissolve the plasmid pWPXLd–hERRα4ug; psPAX23ug; After uniformity, transfer it into the dor...

[0188] This example 3 provides a method for determining the virus titer of the 293T cells transfected with pWPXLd-hERRα provided in Example 2, including the following steps:

[0189] a. Dilute the well-growing 293T cells to 1×10 after digestion and counting 5 / ml, add to 96-well plate, 100ul / well, prepare 10 wells for each virus, place at 37℃, 5%CO 2 Incubate overnight in an incubator.

[0190] b. After 24 hours, the virus was serially diluted 10 times, and 12 serial dilutions were performed.

[0191] c. Discard the original culture medium of the 96-well plate, add the diluted virus solution, and set a blank control. Polybrene (polybrene, working concentration is 6ug / ml) was added to the culture medium to promote virus infection of cells.

[0192] d. Add 100ul culture solution to each well after 48h.

[0193] e. On the fifth day of culture, observe the results under a fluorescent microscope, and count the number of fluorescent cell clones in the two lowest dilution wells w...