Construction method for HA-VP2 gene recombination baculovirus expression vector
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A technology of baculovirus and gene recombination, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problem of low expression rate of foreign genes
Inactive Publication Date: 2012-08-01
HEILONGJIANG UNIV
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[0003] The present invention aims to solve the problem of low expression rate of exogenous genes mediated by baculovirus in CHO cells, and provides a method for constructing HA-VP2 gene recombinant baculovirus expression vector
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specific Embodiment approach 1
[0015] Specific embodiment one: the construction method of the HA-VP2 gene recombinant baculovirus expression vector of the present embodiment is carried out according to the following steps:
[0016] 1. Using the plasmid pMD18-T-VP2 as a template and TZF-1 and TZF-2 as primers, PCR amplification was carried out, the PCR product was subjected to 1% agarose gel electrophoresis detection, and the gel recovery kit was used for purification and recovery to obtain The target fragment, and then carry out TA cloning of the target fragment, and then connect the obtained product to the pMD18-T vector, and obtain the plasmid pTZF-HA-VP2 after the ligation product is identified;
[0017] 2. Plasmids pWK, pWK-I, pLM, pLM-I, pSD, pSD-I, pWKC, pLMC, pSDC and pTZF-HA-VP2 were digested with EcoR I / Sal I respectively, and the target fragments obtained after digestion pWK, pWK-I, pLM, pLM-I, pSD, pSD-I, pWKC, pLMC, and pSDC were respectively ligated with pTZF-HA-VP2 after enzyme digestion with ...
specific Embodiment approach 2
[0073] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that the reaction system of PCR amplification in step 1 is a 50 μL reaction system, which consists of the following components:
[0074]
[0075] PCR amplification conditions were: denaturation at 98°C for 4 minutes, denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, extension at 72°C for 1 minute, a total of 30 cycles, extension at 72°C for 5 minutes, and incubation at 4°C. Others are the same as in the first embodiment.
specific Embodiment approach 3
[0076] Specific embodiment 3: The difference between this embodiment and specific embodiment 1 is that the reaction system for TA cloning of the target fragment in step 1 is 25 μL and consists of the following components:
[0077]
[0078] The TA cloning condition of the target fragment is 72°C for 15 minutes. Others are the same as in the first embodiment.
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Abstract
The invention discloses a construction method for a gene recombination baculovirus expression vector and relates to a construction method for an HA-VP2 gene recombination baculovirus expression vector. The invention aims to solve the problem that the expression rate of a traditional baculovirus-mediated exogenous gene in a Chinese Hamster Ovary (CHO) cell is low. The method comprises the following steps of: I-, performing polymerase chain reaction (PCR) amplification by taking pMD18-T-VP2 as a template to obtain a target fragment, and performing TA cloning and vector conjugation on the target fragment to obtain pTZF-HA-VP2; II-, respectively performing double-enzyme digestion on nine plasmids and the pTZF-HA-VP2, and connecting target fragments obtained after the enzyme digestion is ended to obtain nine recombination transfer vectors; III-, respectively transforming the nine recombination transfer vectors into competent cells, and extracting to obtain rBac-TZF-X; and IV-, performing transfection on the sf9 cells by the rBac-TZF-X to obtain rBV-TZF-X. According to the construction method for the HA-VP2 gene recombination baculovirus expression vector, the expression rate of the baculovirus-mediated exogenous gene in the CHO cell can be increased through adding a regulatory element WPRE.
Description
technical field [0001] The invention relates to a method for constructing gene recombinant baculovirus expression vector. Background technique [0002] Infectious bursal disease is one of the major infectious diseases that endanger the poultry industry in the world. At present, the prevention and treatment of infectious bursal disease is still based on traditional vaccines. However, due to the continuous mutation of virus antigens, especially the emergence of super-virulent strains, the situation of prevention and control of this disease is more severe, and the existing rodents The expression rate of exogenous genes mediated by virus was low in CHO cells. Therefore, it is particularly important to develop new and efficient genetically engineered vaccines to solve the drawbacks of traditional vaccines. Studies suggest that VP2 protein is the main structural protein of infectious bursal disease virus (IBDV) and also the protective antigen of the host. The application of imp...
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