输卵管特异表达人溶菌酶基因重组禽腺联病毒的构建及鉴定

doi:10.16431/j.cnki.1671-7236.2017.10.007

Abstract

Abstract:

In order to produce recombinant avian adeno-associated virus (rAAAV) oviduct-specific expressing human lysozyme (hLYZ) by the baculovirus expression system, one pair of primers was designed according to the published sequences for hLYZ, the hLYZ gene was amplified by PCR and cloned into baculovirus expression vector, which contained the oviduct-specific expression cassette and the inverted terminal repeats of avian adeno-associated virus (AAAV), the resulted plasmid was named as pFB-AIOVLYZ.Then the recombinant vector pFB-AIOVLYZ was transformed into E.coli DH10Bac, and the positive recombinant bacmid rBacmid-AIOVLYZ was screened according to the resistant and the blue-white plague screening,rBacmid-AIOVLYZ was transfected into the Sf9 insect cells by liposome. Once the cytopathic effect was found, the rBac-AIOVLYZ could be harvested.Sf9 insect cells cultured in suspension culture were infected with three recombinant baculoviruses, rBac-AIOVLYZ, rBac-VP and rBac-Rep, at an MOI of 5. 72 h later, Sf9 insect cells were collected, and recombinant viral particles rAAAV-OVLYZ were purified by filtration, chloroform extraction and PEG precipitation. Electron microscopy showed a typical morphologic feature of Parvoviridae family with virus particle size of about 20 nm. PCR results indicated that the target gene existed in the viral genome. The in vitro cell expression test showed that rAAAV could mediate the specific expression of hLYZ in oviduct cells. These results indicated that the rAAAV oviduct-specific expressing hLYZ was successfully prepared by baculovirus expression system, which laid the foundation for the preparation of hLYZ.

Key words: recombinant avian adeno-associated virus (rAAAV); human lysozyme (hLYZ); baculovirus expression system; identification

CLC Number:

Q819

WU Zhi, ZHANG Ji-ling, WU Meng, CUI Xiao-ting, WANG An-ping. Construction and Identification of Recombinant Avian Adeno-associated Virus Oviduct-specific Expressing Human Lysozyme Gene[J]. , 2017, 44(10): 2871-2877.

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Construction and Identification of Recombinant Avian Adeno-associated Virus Oviduct-specific Expressing Human Lysozyme Gene

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