Avian adenovirus transfer carrier and preparation method thereof

A technology for transferring vectors and avian adenoviruses, applied in the field of genetic engineering, can solve problems such as low recombination rate, and achieve the effect of wide application value

Inactive Publication Date: 2013-04-24
SHANGHAI RES CENT FOR MODEL ORGANISMS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has been adopted by many laboratories to prepare recombinant adenovirus, but it also has the problem of low recombination rate, and there may be false positives (non-specific recombination), which requires repeated identification

Method used

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  • Avian adenovirus transfer carrier and preparation method thereof
  • Avian adenovirus transfer carrier and preparation method thereof
  • Avian adenovirus transfer carrier and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0066] Example 1 Type I avian adenovirus AV208 strain

[0067] 1 Materials and methods

[0068] 1.1 Materials

[0069] Type Ⅰ avian adenovirus AV208 strain was purchased from China Veterinary Drug Administration, and its TCID50 on chicken embryonic kidney cells (CEK) was 105.5 / 0.5mL. Chicken liver cancer cell line (LMH) was obtained from the Key Laboratory of Medical Molecular Virology, Ministry of Education, Fudan University. DMEM / F12 culture medium, fetal bovine serum, antibiotics, and trypsin were all purchased from GIBCO Company. Low melting point agarose was purchased from PROMEGA.

[0070] 1.2 Culture and morphological observation of LMH cells

[0071] LMH cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 100 U / mL penicillin and streptomycin, and the cell morphology was observed. In order to explore the culture conditions of this cell line, LMH cells were subcultured at the ratio of 1:3, 1:4, 1:5, and 1:6, and cultured with serum concentra...

Embodiment 2

[0098] Example 2 Construction of Transfer Vector pUC-LR-eGFP

[0099] 1 Materials and methods

[0100] 1.1 Materials

[0101] Type Ⅰ avian adenovirus AV208 strain was purchased from China Veterinary Drug Control Institute. Escherichia coli competent cells DH5α, vectors pMD18-T and pUC18 were purchased from Dalian Bao Biological Engineering Co., Ltd. The pEGFP-N1 plasmid was purchased from Clontech Company. Taq enzyme, T4 DNA ligase, and restriction endonuclease were all purchased from Dalian Bao Biological Engineering Co., Ltd., and Pfu enzyme was purchased from PROMEGA Company. Viral genome extraction kit was purchased from TIANGEN Company. DNA gel recovery kit and plasmid mini-extraction kit were purchased from OMEGA Company.

[0102] 1.2 Extraction of avian adenovirus genome

[0103] Avian adenoviruses were obtained from LMH cells infected with FAV-I AV208. After LMH cells have obvious CPE, digest the cells and blow them off with 1ml of medium, harvest them, freeze a...

Embodiment 3

[0226] Construction and screening of embodiment 3 recombinant virus rFAV-I-eGFP

[0227] 1 Materials and methods

[0228] 1.1 Type I avian adenovirus AV208 strain, purchased from China Veterinary Drug Administration, TCID on chicken embryonic kidney cells (CEK) 50 for 10 5.5 / 0.5mL. Chicken liver cancer cell line (LMH) was obtained from the Key Laboratory of Medical Molecular Virology, Ministry of Education, Fudan University. DMEM / F12 culture medium, fetal bovine serum, antibiotics, and trypsin were all purchased from GIBCO Company. Low melting point agarose was purchased from PROMEGA. The endotoxin-free plasmid mini-extraction kit was purchased from OMEGA Corporation. Lipfectamine 2000, a liposome transfection reagent, was purchased from INVITROGEN.

[0229] 1.2 Massive extraction of transfection-grade plasmid pUC-LR-eGFP Take 30ul of pUC-LR-eGFP transformed Escherichia coli DH5αglycerobacteria stored at -20℃ and inoculate 3ml of fresh ampicillin-containing LB culture m...

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Abstract

The invention discloses an avian adenovirus transfer carrier. PUC18 is regarded as a skeleton plasmid; left and right flanking sequences of an FAVI-AV208 strain genome unnecessary region are inserted in the PUC18 carrier for homologous recombination, but a replicative unessential region of 2980 bp between the left and right flanking sequences is deficient; and a gene expression box including polyA signals of hCMV, MCS, eGFP and SV40 early transcription is inserted between the left and right flanking sequences. The invention also provides a preparation method of the avian adenovirus transfer carrier; and a recombinant virus is also prepared successfully. By introducing a complete foreign gene expression box for one step and adding suitable digestion positions, a carrier construction method is improved, workload is extremely reduced, the replicative unessential region is deleted to a relatively great extent and greater fragmental foreign genes can be contained.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering, and in particular relates to a carrier, in particular to a transfer carrier of avian adenovirus and a preparation method thereof. Background technique: [0002] Adenovirus is one of the most detailed viruses in terms of morphology and chemical structure among animal viruses. The main reason is that adenovirus has no envelope and a clear capsid structure, which is easy to observe carefully under an electron microscope, and nucleic acid and protein are easy to separate. . Moreover, in recent years, the genome structure, replication, and transcription characteristics of some serotypes of adenoviruses have been discovered quite clearly. In particular, the successful clinical application of some adenovirus vectors has made the use of recombinant adenoviruses as vectors to participate in gene therapy and gene immunization highly sought after. General attention. [0003] As a gene transfer vector, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N15/65C12N7/01C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 周洁赵莉朱妍高诚胡建华
Owner SHANGHAI RES CENT FOR MODEL ORGANISMS
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