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Baculovirus vector with long-acting stable expression and construction method thereof

A construction method and recombinant baculovirus technology, applied in the field of baculovirus vector construction, can solve the problems of limited clinical application scope, limited expression level and expression time, and achieve stable expression, improved expression time and expression level. Effect

Inactive Publication Date: 2020-02-04
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the above strategies, the methods of transduction of immune pardon site and in vitro transduction limit the scope of its clinical application, and the potential side effects of pharmacological inhibitors still need to be further evaluated. Complement regulatory proteins such as DAF are transmembrane proteins in Expression levels and expression times in baculovirus are very limited, and chemical modification processing requires a lot of optimization in terms of batch stability and virus infectivity

Method used

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  • Baculovirus vector with long-acting stable expression and construction method thereof
  • Baculovirus vector with long-acting stable expression and construction method thereof
  • Baculovirus vector with long-acting stable expression and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 The construction method and functional verification of the baculovirus vector capable of long-term and stable expression

[0060] (1) Construction of recombinant plasmids

[0061] 1. Construction of pBacSC-CE plasmid

[0062] Design primers SC-F / SC-R to amplify the SC fragment from the plasmid PUC57-SC (preserved in this laboratory, that is, transfer the SC fragment shown in SEQID NO.1 into the PUC57 plasmid), namely GP64 SP-His6- MCS-GP64 TM-GP64 CTD expression element, then insert the element into plasmid pFastBac TM In the corresponding site downstream of the DUAL (Invitrogen) p10 promoter, a CMV-EGFP fragment was inserted downstream of the plasmid pPh promoter, and the fragment was obtained by double enzyme digestion from the plasmid pEGFP-C1 (Clontech). The correct plasmid identified by PCR and sequencing was named pBacSC-CE.

[0063] The relevant primer sequences, restriction sites, amplified fragments and sizes are shown in Table 1, and the identific...

Embodiment 2

[0098] Example 2 Preliminary application of baculovirus vector capable of long-term and stable expression in anti-angiogenic gene therapy of liver cancer

[0099] Considering that the development and metastasis of primary tumors critically rely on the formation of new blood vessels to provide oxygen and nutrients. Therefore, inhibiting angiogenesis has become a very attractive anticancer strategy. The fusion protein (hEA) composed of human endostatin (Endostatin) and angiostatin (angiostatin) showed greater anti-angiogenic activity and stronger anti-tumor efficiency in animal tumor models. In view of this, the present invention uses a long-acting, stably expressed baculovirus vector as a platform to construct a recombinant baculovirus expressing hEA protein, and evaluates its function and anti-tumor effect against liver cancer tumors. It is characterized in that comprising the steps of:

[0100] (1) Construction of recombinant baculovirus expressing hEA protein

[0101] 1. ...

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Abstract

The invention relates to a recombinant baculovirus vector and a construction method thereof. The recombinant baculovirus vector comprises a SB transposon and a gene encoding a decay acceleration factor (DAF). The construction method includes a step of cloning the SB transposon and the gene encoding the decay acceleration factor (DAF) to a baculovirus vector. A recombinant baculovirus vector expression system constructed by the invention can achieve long-acting stable expression of heterologous protein. hEA (fusion protein of endostatin and angiostatin) is expressed in mice through the recombinant baculovirus vector expression system constructed by the invention, thereby significantly improving life and survival rate of tumor-bearing mice. The recombinant baculovirus vector expression system of the invention can effectively protect against attack of a serum complement system, and simultaneously express fluorescent proteins such as EGFP by means of a SB transposition system, facilitatingto continuously observe fluorescence performance in mammalian cells.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a baculovirus vector capable of long-term and stable expression of foreign genes. Background technique [0002] In recent years, the use of baculovirus as a gene delivery vector for gene therapy has become a research hotspot. Compared with other viruses such as adenovirus, adeno-associated virus, and lentivirus, baculovirus has unique advantages. For example, it does not cause disease to humans and mammals, and can be operated in Class I biosafety laboratories; it can replicate in mammalian cells Defects, no pre-existing antibodies; low cytotoxicity; can accommodate the insertion of large fragments of foreign genes; the construction and purification of recombinant viruses are simple. In addition, in addition to effective gene delivery, baculovirus has also been proven to stimulate mammalian cells to produce antiviral immune responses, resist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66C12N7/01A61K48/00
CPCA61K48/0025C07K14/78C07K2319/21C07K2319/23C07K2319/41C12N15/66C12N15/86C12N2710/14043
Inventor 王志昇杨萌萌李梦婷姬勇敢杨文陈健茂
Owner NINGXIA MEDICAL UNIV