Porcine parvovirus infectious cloning system stably carrying genetic marker, construction method and application thereof

Abstract

The invention discloses a porcine parvovirus infectious cloning system stably carrying a genetic marker, a construction method and an application thereof. The blunt-end enzyme sites are introduced into an ITR sequence at both ends of a porcine parvovirus (PPV) genome, and an In-Fusion technology is used, and a non-sense mutation is introduced into a PPV intermediate sequence to obtain a PPV full-length infectious cloning plasmid carrying genetic marker and distinguished with a wild strain. The infectiou cloning plasmid constructed by a reverse heredity operation technology is used for transfection of PK15 cells in vitro, can induce the cells to generate the same cytopathic effect as the parental strain after continuous blind passage, and a rescue virus that having a similar growth trend asthe parent strain and carrying genetic marker can be rescued. The cloning system can quickly and conveniently perform base mutations at any position in the genome, and provides a convenient and effective tool for the subsequent development of attenuated vaccines and multiple vaccines of porcine parvovirus.

Description

technical field [0001] The invention relates to the transformation of virus genome level by using reverse genetic manipulation technology, in particular to the construction of full-length infectious clones of porcine parvovirus carrying genetic markers. Background technique [0002] Porcine parvovirus (Porcine Parvovirus, PPV) is one of the important pathogens that cause reproductive disorders in sows. It can pass through the placental barrier of sows, causing weak fetuses, stillbirths and mummified fetuses in priminatal sows. It is prevalent in various regions , has brought huge economic losses to the global pig industry, but there is no effective prevention and control measures at present. PPV is a member of the genus Parvovirus in the family Parvoviridae. Its genome is a single-stranded negative-strand DNA with a size of about 5000 bp. There are palindromic hairpin structures at both ends of the genome. The 102 bp palindromic sequence at the 3' end is folded to form a Y-s...

AI Technical Summary

Problems solved by technology

PPV is a single-stranded negative-strand DNA virus with a typical terminal repeat (ITR) palindromic sequence at both ends, and this sequence cannot be directly amplified by PCR, so its genome is studied at the overall molecular level It is very difficult, and the current research on its pathogenic mechanism is still not very clear, and the role of each protein in pathogenicity is even less clear. The successful construction of a full-length infectious clone of the viral genome will provide a practical solution to this problem. the solution

At present, a full-length infectious clone of a attenuated PPV strain NADL-2 is obtained abroad by introducing restriction sites for traditional restriction enzyme connection. This method is time-consuming, labor-intensive, and has a long construction period, and cannot be infected with wild strains. Distinguish, and bring great difficulties to the virus transformation from the virus genome level in the later stage

It has been reported that multiple backbone vectors are used to clone viral genome fragments, and infectious clones are obtained through cumbersome steps such as the introduction of restriction enzyme sites. The operation steps are many, the cycle is long, and the construction efficiency is low, which is not conducive to artificial modification of the viral genome.

Images