Double-expression and small interfering RNA replication-defective hepatitis B virus carrier as well as preparation method and application thereof
A hepatitis B virus, small interference technology, applied in the medical field, can solve the problem of shRNA can not target the carrier and so on
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Embodiment 1
[0098] Example 1 A replication-deficient hepatitis B virus vector expressing small interfering RNA, its preparation method and application
[0099] A replication-defective hepatitis B virus vector expressing small interfering RNA, which is genetically modified with the plasmid pCH-9 / 3093 expressing wild-type HBV, to construct a HBV-shRNA dual expression vector: first, through point mutation, in Add a stop codon after the start codon ATG of each protein in the genome; secondly, delete the sequences at positions 203-407 in the C gene region and 1715-1961 in the S gene region; finally, insert between positions 203-407 in the C gene region H1-shRNA expression cassette; U6-shRNA expression cassette was inserted between positions 1715-1961 of the S gene region to construct a replication-deficient hepatitis B virus vector expressing small interfering RNAs for expressing small interfering RNAs targeting hrGFP , whose sequence is as follows:
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Embodiment 2
[0199] Example 2 Replication-deficient HBV vector inhibits expression of hrGFP in liver cancer cell lines
[0200] The replication-deficient HBV vector expressing shG is transfected into the liver cancer cell line HepG2; in addition, it lacks the self-replication ability, and only with the help of the resident virus, new vector particles can be produced and spread, and when the wild-type HBV disappears, the vector spreads Disperse and stop.
[0201] 1. Replication-deficient HBV vectors double express shG to inhibit the expression of hrGFP in liver cancer cells
[0202] 1.1. Cell culture: liver cancer cell line HepG2;
[0203] 1.2. Transfected cells: divided into 3 groups, the transfection reagent is Fugene6 HD (Roche);
[0204] The first group of cells: vectors pCH-203H1-shG, pCH-203U6-shG, pCH-1715U6-shG, pCH-203H1-shG-1715-U6-shG and pCH-9 / 3142 co-transfected double cells, with pCH-9 / 3093 as the control;
[0205] The second group of cells: the vectors pCH-203H1-shG, pCH-...
Embodiment 3
[0210] Example 3 Replication-defective HBV vector double-expression anti-HBV shRNA inhibits HBV replication and expression in liver cancer cell lines
[0211] HBV vectors expressing anti-HBV shRNA and pCH-9 / 3093 were co-transfected into HepG2 cells, and the effects of shRNA vectors on the replication and expression of wild-type HBV were observed, including: detection of cytoplasmic nucleocapsid DNA by HBV full-sequence probe Southern blotting, Then use the replaced C or S gene fragment sequence or the inserted H1 or U6 promoter sequence-specific probes to perform Southern blotting again, and conduct semi-quantitative analysis of the band signal intensity; Western blotting to detect HBsAg in the cell lysate , HBcAg, alpha-fetoprotein, albumin, and tubulin; and PCR detection of HBV DNA in viral particles with envelopes outside cells by PCR with primers across the shRNA expression frame, to prove the replication ability of the vector in the presence of wild-type HBV.
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