Porcine reproductive and respiratory syndrome resisting Marc-145 cell line as well as preparation method and application thereof

Abstract

The invention discloses a porcine reproductive and respiratory syndrome resisting Marc-145 cell line as well as a preparation method and application thereof. The porcine reproductive and respiratory syndrome resisting Marc-145 cell line is obtained by knocking off CD163 extracellular domain SRCR5 of the Marc-145 cell line. The invention further provides gRNA (guide Ribonucleic Acid) for splicing of a pair of shearing Marc-145 cell CD163 receptors SRCR5 and sequences are shown as SEQ ID No. 2 and SEQ ID No. 3 respectively. After an the fifth extracellular fifth structure domain SRCR5 of the Marc-145 cell surface CD163 receptor on the surface of a Marc-145 cell is deleted through a CRISPR / CAS9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) gene editing technology, the obtained porcine reproductive and respiratory syndrome resisting Marc-145 cell line can completely resist PRRSV (Porcine Reproductive and Respiratory Syndrome virus) infection and resist high-pathogenicity virulent strain HP-PRRSV. Moreover, the CD163 receptor on the surface of the porcine reproductive and respiratory syndrome resisting Marc-145 cell line surface CD163 receptor is normally expressed and other biological functions are normal. The porcine reproductive and respiratory syndrome resisting Marc-145 cell line disclosed by the invention has important meanings for inresearching PRRSV pathogenesis and mining novel disease-resisting or susceptible genes; a novel model is provided for PRRSV researches and the porcine reproductive and respiratory syndrome resisting Marc-145 cell line has important guidance meanings on in screening and breeding of PRRSV-resisting pigs.

Description

technical field [0001] The invention belongs to the technical field of cell genetic engineering. More specifically, it relates to an anti-PRRS Marc-145 cell line and its preparation method and application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). PRRS mainly causes abortion and stillbirth in pregnant sows , mummified fetuses, weak piglets, and pigs of all ages, especially piglets, have respiratory symptoms. The characteristic lesion is interstitial pneumonia, and the mortality rate is extremely high. It is a highly contagious global important infectious disease. The disease first broke out in the United States in 1987, and then spread to Europe. The virus was isolated in my country in 1996. Currently, PRRSV is divided into two genotypes based on genome sequence and antigenic...

AI Technical Summary

Problems solved by technology

In 2006, pig high fever broke out in my country, which caused serious economic losses to the pig industry. Later, this strain was defined as a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). At present, Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) is one of the most damaging diseases to the pig industry. Due to the characteristics of long-term maintenance of PRRS, there are no good vaccines and drugs to prevent and control PRRS at present.

PRRSV is difficult to prevent and control mainly in the following aspects: (1) macrophage-loving and immunosuppressive diseases, PRRSV mainly infects alveolar macrophages (Porcinealveolar macrophages, PAMs) of pigs, and PAMs are immune cells that destroy PAMs, thereby Destroy the body's immune system, thereby causing immunosuppression; (2) antigenic variability. At present, PRRSV mutates rapidly, and the use of attenuated vaccines is a reason for the virus to mutate. Recently, it has been reported in the literature that a new strain of PRRSV, NADC30, has appeared in the United States. In China, a new strain similar to that of the United States was also isolated, named NADC30-like, and another literature reported that a PRRSV-causing virus strain with a high degree of homology to the vaccine virus genome was isolated from a pig farm, and its virulence was enhanced. Analysis may be possible (3) The vaccine has no cross-protection, and PRRSV vaccines on the market have almost no cross-protection, and there is no cross-protection between different strains; (4) Antibody dependence is enhanced, and the PRRSV Infection can stimulate the body to produce antibodies, but low-titer antibodies not only cannot neutralize the virus, but promote the proliferation of the virus; (5) persistent virus infection, after PRRSV infection, can detect viral blood in pigs for a long time (6) Mixed infection, mixed infection of PRRSV and other diseases is common clinically at present, especially circovirus, Haemophilus parasuis, porcine pneumonia, etc. and PRRSV The mixed infection of PRRSV makes the prevention and control of PRRSV even more difficult

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