Application of bifunctional vector in inhibiting HBV (hepatitis B virus) infection

A dual-function, carrier technology, applied in the field of genetic engineering

Pending Publication Date: 2018-07-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the targeted therapy of this immune checkpoint molecule using

Method used

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  • Application of bifunctional vector in inhibiting HBV (hepatitis B virus) infection
  • Application of bifunctional vector in inhibiting HBV (hepatitis B virus) infection
  • Application of bifunctional vector in inhibiting HBV (hepatitis B virus) infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Screening of the specific silencing siRNA sequence of the PD-L1 gene of human origin

[0037] A specific silencing siRNA targeting the human PD-L1 gene is designed for the sequence of the human PD-L1 gene, and the sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0038] Using cationic liposome Lipofectamine2000 (purchased from invitrogen), the designed siRNA was transfected into human glioma cell line U251 cells expressing a higher level of PD-L1, and the expression level of PD-L1 gene was detected by qPCR , see the result figure 1 shown. Depend on figure 1 It can be seen that the siRNA sequence shown in SEQ ID NO.1 has the best silencing effect, so the PD-L1 siRNA sequence shown in SEQ ID NO.1 was screened for further functional research.

Embodiment 2

[0039] Embodiment 2 A kind of HBx siRNA sequence

[0040] The nucleotide sequence of the HBx siRNA sequence is shown in SEQ ID NO.4.

[0041] The synthetic HBx siRNA was transfected into HBV-infected human liver cell line HepG2.2.15 cells using cationic liposome Lipofectamine2000, and the expression levels of HBx gene and protein were detected by qPCR and Western Blot techniques. The results were as follows: figure 2 shown. Depend on figure 2 It can be seen that the siRNA sequence has a good silencing effect on the expression of HBx gene and protein.

Embodiment 3

[0042] Example 3 A method for constructing an RNAi bifunctional carrier targeting PD-L1 and HBx

[0043] The construction method of the RNAi bifunctional carrier targeting PD-L1 and HBx comprises the following steps:

[0044] (1) Connect the PD-L1 siRNA with the nucleotide sequence shown in SEQ ID NO.1 and the HBx siRNA sequence with the nucleotide sequence shown in SEQ ID NO.4 in series to grow a hairpin structure, and add dual enzymes at both ends of the sequence Cutting site BamH I and EcoRI sequences, annealing to form lhRNA double-stranded structure, h-shPD-L1-shHBx-F nucleotide sequence shown in SEQ ID NO.5; h-shPD-L1-shHBx-R nucleotide The sequence is shown in SEQ ID NO.6;

[0045] (2) The pSIREN vector is subjected to BamH I and EcoRI double enzyme digestion, and the vector backbone is recovered to obtain the pSIREN linear vector;

[0046] (3) Ligate the tandem lhRNA prepared in step (1) with the pSIREN linear vector prepared in (2) to construct the pSIREN-shPD-L1-sh...

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Abstract

The invention belongs to the technical field of gene engineering and particularly relates to an application of a dual expression vector of silent RNA (ribonucleic acid) of PD-L1 and HBx target genes in inhibiting HBV (hepatitis B virus) infection. Compared with a monofunctional vector, pSIREN-shPD-L1-shHB constructed has a better inhibition effect on HepG2.2.15 cell proliferation and a better promotion effect on cell apoptosis; the detection of an intracellular anti-apoptosis protein expression situation via Western Blot finds that a bifunctional vector group can obviously reduce expression ofBcl-2 and Bcl-xl; and the detection of an intracellular anti-apoptosis protein gene expression situation via quantitative PCR (polymerase chain reaction) finds that the bifunctional vector group canobviously reduce mRNA (messenger ribonucleic acid) expression of Bcl-2 and Bcl-xl.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of dual expression vectors of PD-L1 and HBx target gene silencing RNA in inhibiting HBV infection. Background technique [0002] Hepatitis B virus (HBV), first identified in 1966, is a hepatropic DNA virus mainly transmitted through body fluids such as the digestive tract or blood. Chronic HBV infection is an important cause of hepatitis, liver cirrhosis and liver cancer. [0003] RNA interference (RNA interference, RNAi) refers to the phenomenon that small non-coding RNA induces mRNA degradation, translation inhibition or promotes heterochromatin formation, etc., and then silences gene expression, resulting in the corresponding loss of functional phenotype. The birth of this technology enables people to efficiently and specifically silence viral mRNA according to their own needs, thereby inhibiting the production of viral antigens and pro...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66A61K31/7088A61P1/16A61P31/20
CPCA61K31/7088C12N15/66C12N15/85
Inventor 张建赵荣荣韩秋菊
Owner SHANDONG UNIV
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