KMD Bioscience Co., Ltd.
400-621-6806
2023-08-08 Hits(410)
The insect expression system is a method to obtain the recombinant virus by transposing the expression components in the transfer vector to the baculovirus shuttle vector proliferated in E. coli, extracting the DNA of the shuttle plasmid and transfecting the insect cells to obtain the offspring of the virus. Bacmid is a baculovirus shuttle vector, which is a method of infiltrating virus supernatant into insect cells to obtain expressed recombinant proteins.
First, we obtained the target gene by PCR amplification.
Construct the target gene into the transfer vector pFastBac.
DH10Bac strain is the host cell of the pFastBac vector. pFastBac vector has a Tn7 transposable element, which can integrate the target gene into the specific site on the Bacmid plasmid of E. coli by transposition, and the insertion of the target gene will lead to the shift of the LacZ gene on the bacmid, which will lead to the identification of a positive recombinant bacmid through the blue-white spotting screening. The bacmid was extracted and transfected into insect cells.
White monoclonal colonies were picked and inoculated with 5 ml of bacteria containing 50 mg/ml kanamycin, 7 mg/ml gentamicin, 10 mg/ml tetracycline, shaking the bacteria, and the bacterial liquid PCR was used for preliminary screening of positive clones.
A small amount of plasmid was extracted and PCR verified the correctness of the pole plasmid.PCR verified correct recombinant plasmid can be used for subsequent transfection of insect cells to obtain the P1 and P2 generation viruses as well as the final target proteins. The plasmid was extracted in small amounts and PCR verified the correctness of the pole plasmid.
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Figure 1: Construction Experiment Process of Bacmid
KMD Bioscience has a perfect insect expression and purification system. The insect cell protein expression system is suitable for recombinant expression of membrane proteins, and 4 times and below transmembrane proteins can be almost effectively expressed and purified in insect cells. Purification of membrane proteins is time-consuming and labor-intensive. KMD Bioscience can provide customized products of full-length membrane proteins with purity of up to 80%. KMD Bioscience can provide customers with a complete set of protein expression solutions, free of charge for the sequence analysis of the genes provided by customers; at the same time, according to the codon preference of Sf9, Sf21, Hi-5 and S2 insect cells, free of charge for you to optimize the codon, to effectively improve the expression of recombinant proteins, to provide you with one-stop service.
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