41 results about "HBx" patented technology

The invention provides a self-processing RNA expression cassette which includes at least one pair of processing units, an RNAi effecter sequence of predetermined length that regulates target gene expression which is flanked by said pair of processing units; and at least one pair of cognate ribozyme cis-cleavage target sites located 5′ and 3′ of the RNAi effecter sequence. The self-processing RNA expression cassette is able to express in vivo and in vitro and the RNAi effecter sequence includes at least one target recognition sequence derived from the Hepatitis B Virus (HBV) X gene (HBx).

The invention discloses a method for constructing a dual-color fluorescence reporter vector and application thereof. The sequence of the dual-color fluorescence reporter vector is a nucleotide sequence as shown in SEQ ID NO.1. The method for constructing the dual-color fluorescence reporter vector comprises the following steps of: A. the construction of derived plasmid pEGFP-N1-Ampicillin; B. the construction of derived plasmid pMD18-EGFP; C. the construction of derived plasmid pMD18-EGFP-polyA; D. the construction of derived plasmid pEGFP / EGFP; E. the final construction of target plasmid pEGFP / DsRed; F. the preparation of a derived dual-color fluorescence vector pEGFP / DsRed-HBx of the dual-color fluorescence reporter vector pEGFP / DsRed. The method has the advantages of easy implementation and convenient operation, and the vector can be applied to high throughput screening and quantitative siRNA efficiency.

The invention belongs to the field of gene therapy and aims at providing a novel targeted gene vaccine for treating HBV (Hepatitis B Virus)-relevant liver cancer and application of the novel targeted gene vaccine in preparation of a targeted gene immunotherapeutic drug for the HBV-relevant liver cancer. The main active component of the liver caner-resistant vaccine disclosed by the invention is a recombinant vector of a gene coded with an HBx protein and a gene for coding a human IL-12 protein; and the recombinant vector can be used for simultaneously expressing the HBx protein and the human IL-12 protein in a eukaryotic cell. Provided by the experiment, a recombinant adenovirus vaccine disclosed by the invention can be used for selectively killing HBx-electropositive liver cancer cell and expresses a favorable liver cancer-resisting effect. The recombinant adenovirus vaccine provides a new selection for targeted gene immunization therapy of the HBV-relevant liver cancer and has a favorable application prospect.

The invention relates to an siRNA (small interfering RNA) directed at HBx and a liposome for transferring the siRNA. The liposome contains siRNA plasmids, cationic lipid 98N12-5, cholesterol and PEGylated phospholipid. The liposome not only has good in vitro-in vivo stability, can well reach the target part, but also acquires high transfection efficiency, thus being in favor of playing a role in conveying siRNA into cells to inhibit hepatitis B virus gene HBx expression. In addition, the liposome also has low toxicity, thus being beneficial to its application in-vivo study.

The invention discloses a Stat3 target CRISPR / Cas carrier, a Stat3-HBx dual-target CRISPR / Cas carrier, and two repeated-target CRISPR / Cas carriers targeting HBx, and particularly relates to an Stat3 target single-function plasmid, an Stat3-HBx dual-target CRISPR / Cas carrier, two CRISPR / Cas carriers repeatedly targeting the HBx, and a construction method of the carriers. Through tests, the inventorproves that the constructed carriers have a specific target treatment effect on HBV-related liver cancer cells, and the silent gene carrier, the carrier construction method, double-silence strategiesof the carriers for Stat3 and the HBx and application of the carriers to preparation of HBV chronic infection and anti-liver cancer related drugs are within the scope of right protection correspondingly.

The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target HBx mediated activation of Src kinase, members of the Src tyrosine kinase family and components of the Src kinase family signal transduction pathways for the treatment of HBV infection and related disorders and diseases, such as HCC. The invention further relates to pharmaceutical compositions for the treatment of HBV infection targeted to HBx and its essential activities required to sustain HBV replication. The invention is based, in part, on the Applicants' discovery that activation of Src kinase signaling cascades play a fundamental role in mammalian hepadnavirus replication. Applicants have demonstrated that HBx mediates activation of the Src family of kinases and that this activation is a critical function provided by HBx for mammalian hepadnavirus replication.

The invention discloses an artificial transcription factor capable of specifically combining a C2H2 trizinc finger protein and a six-zinc finger protein in a hepatitis B virus (HBx) gene transcription regulation domain and fusing an effect function domain capable of inhibiting gene transcription. The invention further discloses an application of the artificial transcription factor to the preparation of drugs for treating HBx infectious diseases. The transcription factor has an obvious inhibiting effect on the transcription of HBx genes and the growth of Hep3B cells integrated with target genes HBx.

A hepatitis B virus antisense RNA expression vector targeted toward liver cancer is a gene therapeutic matter for hepatitis B virus related liver cell cancer. The present invention adopts EB virus expressing plasmid pEBAF as vector and positive liver cancer tissue expressing vector controlled by AFP promotor controlling and specifically expressed in AFP only to construct three kinds of HBV antisence RNA expression vectors pEBAF-as-preS2, pEBAF-as-preS1 and pEBAF-as-X, which contain HBV preS2, preS1 and X segment genes separately. Antisense RNA expression vector is prepared through amplifying a destination gene, designing, a primer, and a PCR amplification of HBpreS2, HBpreS1 and HBX genes. The antisense RNA expression vector is used for treating hepatitis B virus related liver cell cancer and hepatitis B virus infection.

The invention discloses a method for efficiently expressing an AFP3-PTEN fusion protein, and belongs to the technical field of biology. The method comprises the steps of cloning an HBx gene to a constructed expression vector, enabling the constructed vector to be transfected with human liver cancer cells, after expressing HBx proteins in the liver cancer cells, enabling the third structured domainof AFP and an overall-length PTEN gene to be connected to the expression vector and transfected into the human liver cancer cells expressing the HBx proteins, expressing the HBx proteins in the livercancer cells, promoting efficient secretory expression of a human AFP3-PTEN fusion protein through the HBx proteins, and concentrating and purifying culture mediums to obtain the AFP3-PTEN fusion protein. The AFP3-PTEN fusion protein expressed by the liver cancer cells can restrain cell proliferation and can enable the cell proliferation capacity of NIH 3T3 cells to be reduced by 15%-60%. But a reference protein AFP enables cell proliferation capacity to be obviously increased by 22-125%. Serum proteins do not have notable capacity for promoting NIH 3T3 cell proliferation. The obtained AFP3-PTEN fusion protein can be used for researching tumor treatment.

The invention provides an application of dicoumarol in preparation of an HBx protein stability inhibitor. Experiments find that the HBx protein stability inhibitor, especially dicoumarol, can effectively reduce the stability of HBx, so as to inhibit the replication of HBV virus, achieve the purpose of treatment of hepatitis B, provide a new path for treatment of hepatitis B, and a high positive significance for the health of human beings is achieved.

The invention belongs to the field of a biological technology, and particularly relates to a purification method of an HBx protein core segment HBx delta N delta C in HBV and an HBx delta N delta C monoclonal antibody. A formula and purification process of various buffered solutions in an original purification method are changed, so that HBx delta N delta C samples being active, stable, uniform, high in purity and solitary are obtained, and standard large-scale production and preparation are convenient. The obtained HBx delta N delta C are used for further preparing a mouse sourced HBx delta Ndelta C monoclonal antibody and hybridoma strains which can stably express the mouse sourced HBx delta N delta C monoclonal antibody. The HBx delta Ndelta C and the HBx delta N delta C monoclonal antibody can be used as a scientific research reagent, the mouse sourced HBx delta N delta C monoclonal antibody can also be used as a detection reagent of hepatitis b virus HBV infection diseases, and the mouse sourced HBx delta N delta C monoclonal antibody is humanized, so that humanized HBx delta N delta C monoclonal antibody can be obtained and can be used as a scientific research reagent, a detection reagent for HBV infection diseases and an auxiliary treatment preparation.

The invention relates to nucleic acids, including lentiviral vectors and lentiviral vector particles, encoding at least one Hepatitis B virus (HBV) envelop surface of genotypes A and / or C antigen, at least one polymerase of genotypes A and / or C antigen, at least one HBX protein of genotypes A and / or C antigen, at least one HBV consensus core of genotypes A and / or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and / or C antigen. The invention encompasses these lentiviral vectors and lentiviral vector particles, methods of making the vectors, and their use, including medicinal uses. The lentiviral vectors and lentiviral vector particles are for use in administering to humans to induce immune responses against the HBV antigens.

Provided herein are, inter alia, antibodies, antigen-binding antibody fragments, cells, polynucleotides, compositions, kits, and methods relating to the detection of HBV protein X (HBx), e.g., in vitro and in vivo. Included are antibodies and fragments thereof that bind HBx, as well as kits, cells, and compositions comprising such antibodies and fragments.

The invention belongs to the technical field of biology, and particularly relates to a dissolving and purifying method of an HBx protein inclusion body derived from Hepatitis B Virus HBV. The preparation method comprises the following steps: expressing HBx by using a recombinant expression system; collecting cells containing the HBx protein which has been subjected to recombinant expression, breaking the cells by using a lysis buffer solution under a cooling condition, and collecting a part containing an HBx protein inclusion body; adding a dissolution buffer solution into the obtained part containing the HBx protein inclusion body for resuspension and denaturation; performing renaturationon on the product, removing nucleic acid, performing purification, and obtaining HBx protein; whereinthe steps are completed under the environmental condition of less than or equal to 10 DEG C. By using the method provided by the invention, a stable and uniform monomer HBx sample can be obtained, andthe concentration can reach 15mg / ml. The protein sample can be used as a scientific research reagent, and a monoclonal antibody of HBx can be prepared by using the protein sample.

The invention provides a deletion mutant of hepatitis B HBX gene, wherein SEQ ID NO. 1 is a mutant lack of 264 bases in 1577-1840 HBV nucleic acid position; SEQ ID NO. 2 is a HBX gene mutant lack of 19 bases in 382-400 nucleic acid position; SEQ ID NO. 3 is a HBX gene mutant lack of 15 bases in 431-445 nucleic acid position. The deletion mutant is provided with apparent oncogenic effect, which can be used to the gene detection of hepatitis B etiological agent carrier and hepatic carcinoma.

The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target HBx mediated activation of Src kinase, members of the Src tyrosine kinase family and components of the Src kinase family signal transduction pathways for the treatment of HBV infection and related disorders and diseases, such as HCC. The invention further relates to pharmaceutical compositions for the treatment of HBV infection targeted to HBx and its essential activities required to sustain HBV replication. The invention is based, in part, on the Applicants' discovery that activation of Src kinase signaling cascades play a fundamental role in mammalian hepadnavirus replication. Applicants have demonstrated that HBx mediates activation of the Src family of kinases and that this activation is a critical function provided by HBx for mammalian hepadnavirus replication.

The invention provides application of 3-trametenolic acid B methyl cyanide in preparing anti-hepatitis B virus drugs, and further relates to application of the 3-trametenolic acid B methyl cyanide inpreparing drugs inhibiting HBsAg secretion of a human hepatocellular carcinoma HepG2 / 2.2.15 cell strain, or application of 3-trametenolic acid B methyl cyanide in preparing drugs expressing hepatoma carcinoma cell HBx expression. The 3-trametenolic acid B methyl cyanide has positive effects on preventing hepatitis b virus infection, treating hepatitis B infection, preventing hepatocellular carcinoma, treating hepatitis B related hepatocellular carcinoma and preventing relapse in the whole process of lesions. The great significance on solving the technical problem of clinically treating hepatitis B infection, and especially the hepatitis B related hepatocellular carcinoma is achieved.

The invention discloses a Stat3-targeting CRISPR / Cas carrier, a Stat3-HBx dual-targeting CRISPR / Cas carrier and two repeat-targeting HBx-targeting CRISPR / Cas carriers, in particular to a Stat3-targeting single function Plasmids, a Stat3-HBx dual-targeting CRISPR / Cas vector, two repeating HBx-targeting CRISPR / Cas vectors, and methods for constructing the above-mentioned vectors. The inventors have proved through experiments that the vector constructed in this invention has a specific targeted therapeutic effect on HBV-related liver cancer cells. The present invention correspondingly protects the above-mentioned silencing gene vector, the construction method of the vector, the dual silencing strategy of the vector for Stat3 and HBx, and The application of the carrier in the preparation of HBV chronic infection and anti-liver cancer related drugs.

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.

The invention belongs to the field of biotechnology and genetic engineering, and relates to a method for evaluating an X protein (HBx protein) binding molecule of human hepatitis C virus. The method provided by the invention comprises the following steps: expressing a secreting type fusion protein containing the HBx protein and luciferase NanoLuciferase (Nluc) in a cell; and quantifying the levelof the secreted HBx protein by detecting the activity of luciferase of the fusion protein. The method can be used for screening and identifying the HBx protein binding molecule, provides a support forresearching an HBx protein targeted drug, and has good application prospect.

The invention belongs to the technical field of biological medicines, and particularly discloses application of asiatic acid to preparation of a medicine for treating hepatitis B. It is found that asiatic acid can reduce the HBx protein level and inhibit the transcriptional activity of cccDNA so as to reduce the total HBV RNAs and HBV 3.5-kb RNA levels in cells and the HBsAg level in supernatant, and can effectively reduce the HBsAg and HBV DNA levels in serum and the HBV RNA and HBV DNA levels in liver tissue in a recombinant cccDNA mouse model, and the asiatic acid is a potential novel HBx targeting medicine and can be used for treating hepatitis B. The invention provides a new direction and theoretical basis for reducing the infection rate of HBV and exploring a novel medicine for effectively treating hepatitis B.

Provided herein are, inter alia, antibodies, antigen-binding antibody fragments, cells, polynucleotides, compositions, kits, and methods relating to the detection of HBV protein X (HBx), e.g., in vitro and in vivo. Included are antibodies and fragments thereof that bind HBx, as well as kits, cells, and compositions comprising such antibodies and fragments.

The invention relates to the technical field of nano-drugs, in particular to cationic liposome nanoparticles co-loaded with HBx-siRNA and schisandra chinensis oil as well as a preparation method and application thereof. According to the invention, small interfering RNA (Ribonucleic Acid) aiming at HBx is established, schisandra chinensis oil with a liver protection effect is selected, and water-soluble substances and fat-soluble substances are simultaneously loaded in the cationic liposome by utilizing the characteristic that the cationic liposome can simultaneously load the water-soluble substances and the fat-soluble substances, so that the preparation process is optimized; and the cationic liposome nanoparticles co-loaded with the HBx-siRNA and the schisandra chinensis oil, which are prepared by the optimal process, are subjected to pharmacodynamic research on in-vitro inhibition of hepatitis B. Experiments prove that the schisandra chinensis oil in the cationic liposome nanoparticles has a good effect of cooperating with HBx-siRNA to inhibit hepatitis B.

The invention discloses a method and kit for detecting an HBV genotype and / or X-region mutation, a CDS standard sequence of HBx, a primer and application. The method comprises the following steps: selecting CDS of middle HBx of a sequence and CDS standard sequences of 10 kinds of HBx for homology analysis and comparison to obtain a result of the HBV genotypes and / or X-region mutation to be detected; further, judging whether the HBV genotype and / or X-region mutation is a single genotype or a mixed genotype through the peak nesting condition in a sequencing peak graph, and if no nested peak exists or the number of nested peaks is smaller than 10, determining the HBV genotype and / or X-region mutation as the single genotype; and if the number of the nested peaks is greater than or equal to 10,determining the HBV genotype and / or X-region mutation as the mixed genotype. The invention provides a set of convenient and high-accuracy scheme for simultaneously detecting 10 kinds of HBV genotypesand X-region mutation types, and a reference basis can be provided for HBV infected person disease progress risk assessment, antiviral crowd screening, antiviral mode selection and curative effect prediction.