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Recombined viral vectors and uses thereof

A recombinant viral vector and viral vector technology, applied in the field of recombinants, can solve the problems of weak immune response, carcinogenesis of exogenous nucleic acid, etc.

Active Publication Date: 2008-07-16
恒瑞源正(上海)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therapeutic hepatitis B vaccines that have entered the clinical phase I abroad and are in the laboratory stage at home and abroad are all DNA vaccines. The current problems of DNA vaccines are whether the exogenous nucleic acid will be integrated into the chromosome to cause cancer, and whether it can cause immunopathological effects. There are definite conclusions, and the immune response induced by the vaccine is weak

Method used

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  • Recombined viral vectors and uses thereof
  • Recombined viral vectors and uses thereof
  • Recombined viral vectors and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the construction of CHB4 recombinant adenovirus vaccine

[0042] 1. Acquisition of the internal ribosome entry site (IRES, Internal Ribosome Entry Site) fragment: using the pIRES vector (purchased from BD Company, whose plasmid map is shown in Figure 1) as a template, design primers: IRES(F)5' ACG ACT CAC TAT AGG CTA 3', IRES(R) 5' TCG ACT CTA GAG GAT CC, (Synthesized by Huada Genomics Shanghai Ding'an), for PCR amplification, the amplification conditions are: the first cycle of denaturation at 94°C for 5 minutes , each subsequent cycle: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, a total of 30 cycles, and extension at 72°C for 5 minutes. The IRES fragment was obtained, and its specific sequence is shown in SEQ ID NO:3. The amplified PCR fragment was inserted into a T vector (Promega Company) to obtain a T / I vector, see FIG. 3A .

[0043] 2. Acquisition of the nucleotide sequence HBs encoding the...

Embodiment 2

[0048] Embodiment 2, the construction of CHB5 recombinant adenovirus vector

[0049] 1. Obtaining the IRES2 fragment: artificially synthesize the IRES2 sequence, as shown in SEQ ID NO: 4, and use it as a template to design primers: IRES-MluI(F): 5'ACGCGTTATCCCTTGCGG 3'IRES-SmaI(R): 5' CCCGGGTGTGGCAGGAGT 3' (Shanghai Ding'an Synthetic Co., Ltd. of BGI) was used for PCR amplification. The amplification conditions were: denaturation at 94°C for 2 minutes in the first cycle, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and 72°C for each cycle. Extension was performed for 40 seconds for a total of 30 cycles, followed by a 7 minute extension at 72°C. Obtain the IRES2 fragment. The amplified PCR fragment was inserted into a T vector (Promega Company) to obtain a T / IRES2 vector.

[0050] 2. Acquisition of the adenovirus shuttle vector PDC515 / CIS2: Please refer to Figure 5, digest plasmid T / IRES2 with restriction enzymes M1uI and SmaI to obtain the IRES2 fra...

Embodiment 3

[0052] Embodiment 3, the construction of HB-S'IC' recombinant adenovirus vaccine (CHB6)

[0053] 1. Obtaining the IRES fragment: Obtain the T / I vector by the same steps as in Example 1.

[0054] 2. Obtaining of HBs' fragments: extract the serum of hepatitis B patients, extract hepatitis B virus DNA from the patient's serum, and use the extracted DNA as a template to design primers: HBs-NheI (IF): 5'GCTAGCCAACCATGGAGAGCACA3' (the underline is the enhancer) , HBs-XhoI (R): 5'CTCGAGTCAAATGTATACCCA3' (Synthesized by Huada Genomics Shanghai Ding'an), PCR amplification, amplification conditions: the first cycle of denaturation at 94°C for 5 minutes, subsequent cycles: denaturation at 94°C for 30 seconds , annealing at 61°C for 30 seconds, extension at 72°C for 1 minute, a total of 30 cycles, and then extension at 72°C for 7 minutes. Obtain HBs' fragment. Insert its PCR fragment into the T vector to obtain the T / S' vector.

[0055] 3. Acquisition of HBc' fragments: Extract the ser...

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Abstract

The invention relates to a genetic engineering technology, in particular to a recombinant virus vector. The vector includes the virus vector and at least two nucleotide sequences, the nucleotide sequences respectively code the antigens of pathogenic virus, the antigen comprises a B cell epitope and a T cell epitope; the recombinant virus vector can lead immune animals to produce humoral immune and cell immune reactions, thus achieving the purpose of the prevention and treatment of diseases. The injection of the recombinant adenovirus vector of the invention has high safety at the same time.

Description

technical field [0001] The present invention relates to genetic engineering technology, in particular to a recombinant constructed with nucleotide sequences encoding pathogenic virus antigens and viral vector sequences. Background technique [0002] Hepatitis B is caused by Human Hepatitis B virus (HBV). HBV is a DNA double-stranded virus and is mainly transmitted through blood, close contact and vertical transmission from mother to child. Different regions have different HBV infection rates and modes of transmission. After HBV infection, some patients will develop chronic persistent infection, and some may develop liver cirrhosis or primary hepatocellular carcinoma. At present, it is estimated that there are about 350 million hepatitis B carriers in the world, and about 220 million in Asia. There are about 130 million people in my country who are carriers of hepatitis B virus, and the annual medical expenses for the treatment of hepatitis are about 30-50 billion yuan, and...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/51C12N15/37C12N15/44A61K48/00A61P31/14A61P31/20A61P1/16
Inventor 李进唐云霞秦华蒋英丽向周军
Owner 恒瑞源正(上海)生物科技有限公司
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