Selection marker-free recombinant goat-pox virus for expression of Orf virus F1L protein and construction method thereof

Abstract

Through homologous recombination and by innovative utilization of plaque and reverse plaque screening technologies, the selection marker-free recombinant virus, especially the rGTPV-F1L recombinant goat-pox virus is finally obtained. The method is simple and effective, is easy to operate, provides the selection marker-free recombinant virus for the development of a recombinant virus vaccine, provides new ideas and measures for the researches on a novel ORF vaccine, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a recombinant virus, and more particularly to a recombinant goat pox virus without a selection marker and a construction method thereof. Background technique [0002] Orf, also known as Contagious Ecthyma (CE), is a zoonotic disease caused by Orfvirus (ORFV). One of the most important viral diseases affecting goats and sheep. The disease mainly affects the lips, tail bases and breasts of sheep, and often leads to symptoms such as pustules, ulcers and nodules at the infected site. The disease spreads rapidly, is widespread, and has a high incidence rate. Sheep of different breeds, genders and ages can be infected. The morbidity and mortality rate is the highest in lambs. Due to the good tropism of ORFV on the lips of lambs, lambs are affected. Posterior lip wounds are often secondary to infection with a variety of pathogens, resulting in difficulty in eating, decreased immunity,...

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Problems solved by technology

However, this is still a problem for the industry

Guo Fei et al. from the Institute of Pathogen Biology, Chinese Academy of Medical Sciences (A vaccinia virus vector with high efficiency recombination and no selection marker and its establishment method, CN201910370701.7, 2019-07-19.) used CRISPR-Cas9 technology to knock out vaccinia virus The TK gene is then labeled with eGFP fluorescence on the poxvirus, and then the marker gene is deleted using the Cre-LoxP system. Although the efficiency of homologous recombination has been improved, there are the following disadvantages: (1) CRISPR-Cas9 is an emerging technology in recent years. At this stage, we mainly face problems such as low precision repair ratio, limitation of PAM recognition sequence, off-target phenomenon, mosaic phenomenon, etc. Many aspects still need to be continuously improved and perfected; (2) In the process of constructing unmarked recombinant poxvirus, gene editing After the technology, it still needs to go through multiple plaque purification screenings, which does not simplify the virus purification process, and the purification of recombinant viruses is the key to obtaining recombinant viruses

[0008] CN 102559611A discloses a double-expression recombinant MVA virus without screening markers and its construction method, in which homologous recombination technology and terminal dilution method are used to construct and screen marker-free recombinant viruses, but it has the following disadvantages: First, the selected Ankara The vaccinia virus (MVA) vector cannot effectively proliferate in the human body and most mammalian cells. The use of hamster kidney cells (BHK21) for liposome transfection to construct recombinant MVA virus will inevitably affect the efficiency of homologous recombination, which will affect the construction of recombinant virus. Second, in the process of constructing the recombinant virus, the target gene was not marked, resulting in the inability to visually and effectively distinguish the recombinant virus from the parental virus, making the purification and identification of the recombinant virus very difficult, which is why most of the current research The difficult problem to be overcome in constructing the recombinant virus containing the screening marker gene, and the paper does not give a more effective solution; then, in the purification process of the recombinant virus, the BHK21 cells are replaced by rabbit kidney cells (RK-13), However, the proliferation of viruses on new cell lines requires an adaptation period, which increases the difficulty of virus purification, and the use of endpoint dilution method to obtain recombinant viruses requires a greater workload and is prone to false positives.

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