Corn expression vector of Hepatitis B virus L gene and application thereof
A technology of hepatitis B virus and expression vector, applied in the field of biomedical engineering, can solve the problem of weak HBVS gene variant strain and other problems
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Embodiment 1
[0060] Example 1: The schematic diagram of the construction of the maize expression vector pCAMG-L comprising the hepatitis B virus L gene is as follows figure 1 shown
[0061] (1) Design of cloning gene primers:
[0062] Design 1 pair of primers according to the coding sequence of HBV L gene:
[0063] The upstream primer sequence is: P1 5'-TTATCG GGATCC ATGGGAGGTTGGTCTTC-3', containing BamH I restriction site and initiation codon ATG;
[0064] The downstream primer sequence is: P25'-CGCGCTGCTAGCTCAAATGTATACCCAAAAG-3', containing NheI restriction site and stop codon;
[0065] (2) PCR reaction and cloning:
[0066] The L gene amplification template is taken from the serum of patients with positive hepatitis B surface antigen; the serum is extracted by phenol / chloroform extraction method; the extraction of hepatitis B virus DNA: collect venous blood, mix 100 UL serum with 1 UL proteinase K (PK) buffer, Add 10UlpK (20mg / mL) and leave overnight at 55°C. Mix with 250 UL satura...
Embodiment 2
[0102] Extract and screen the recombinant expression vector pCAMG-L obtained in Example 1, and transform it into Agrobacterium LBA4404. Prepare the competent Agrobacterium LBA4404 in advance and store it in a -80°C refrigerator for later use; take 200 μL of competent cells and place them on ice for 30 minutes; add 1 μg of DNA to the competent In the state cells, quick-freeze in liquid nitrogen for more than 5 minutes; quickly thaw in a water bath at 37°C for less than 5 minutes; add 800 μL LB medium; culture on a shaker at 28°C at low speed for 3 hours; take 200 μL and spread it on a plate containing 50 μg / mL kanamycin, 28 Cultivate on a shaker at ℃ for 2d to 3d; after colony PCR verification, select positive clones and shake the bacteria, extract the plasmid and carry out double enzyme digestion identification, and finally confirm that the recombinant plasmid has been transferred into LBA4404, and store the strain at -80°C (see Figure 5 with Figure 6 ).
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