Effects Of Pig Interferon Induced Protein 44L On Proliferation Of TGEV And Preparation Of Its PcAb

Transmissible gastroenteritis（TGE）is a class B animal disease caused by Transmissible gastroenteritis virus（TGEV）.In recent years,the outbreak of TGE in pig farms has caused great resistance to the development of pig industry.At present,with the global spread of human Novel Coronavirus（2019-n Co V）,the safety of human lives is under serious threat.A growing number of researchers are looking for solutions to effectively combat the coronavirus in humans and animals.Interferon（IFN）has recently become a research hotspot due to its important antiviral effect in the innate immune process of the body.IFN-stimulated genes（ISGs）are among the most important innate host defence mechanisms against viral infection.More and more ISGs have been identified,including Interferon Stimulation gene 15（ISG15）,2’-5’-Oligoadenylate synthetase,2’-5’-OAS,Myxovirus Resistance Protein A（Mx A）,Myxovirus resistance protein B,Mx B,Interferon stimulation gene 44（IFI44）,and Interferon stimulation gene44-like（IFI44L）.Current studies indicate that different ISGs have different regulatory effects and mechanisms on virus proliferation.As IFI44L is a newly identified ISG,little is known about its antiviral effect and mechanism of IFI44L.Therefore,it is of great significance to study the regulatory function and underlying mechanism of IFI44L on virus proliferation.Based on these facts,the porcine IFI44L gene was cloned and sequenced for the first time in this study.Furthermore,SYBR GreenⅠfluorescent quantitative PCR and TCID₅₀were used to explore the effects of IFI44L on TGEV proliferation.Finally,Polyclonal antibody（Pc Ab）against porcine IFI44L protein was prepared,and its specificity and stability were confirmed by Western-blot and Indirect immunofluorescence assay（IFA）.This study sheds light on further investigating the structure and biological functions of IFI44L protein,and provide new ideas and tools for the prevention and control of TGE and other coronavirus diseases.The main contents of this study are as follows:1.Amplification,sequence analysis and phylogenetic tree mapping of pig IFI44L geneAccording to the IFI44L gene m RNA sequence of human in NCBI,proper primers were designed.The total c DNA of porcine kidney cell（PK-15）was employed as the temple to amplify the porcine IFI44L gene sequence.The results showed that the sequence of porcine IFI44L CDs was 1320bp in length which encoded 439 amino acids.The multiple sequence alignment of the amino acid sequences illustrated that pig IFI44Lshared a 63.01%homology with that of human,and 48.97%with that of mouse IFI44L protein,respectively.Phylogenetic trees of pig IFI44L and IFI44L coding regions of different species published in Gen Bank were drawn by MEGAX64 software.Pig IFI44L was found to be the most closely related to that of cattle.2.Effects of porcine IFI44L on proliferation of transmissible gastroenteritis virusThe eukaryotic expression recombinant plasmid p CAGGS-HA-44L was constructed using p CAGGS-HA as a vector.After confirming the high expression level of p CAGGS-HA-44L in PK-15 cells by Western-blot analysis,p CAGGS-HA-44L was transfected into PK-15 cells and then infected with TGEV with different MOI 24 h post transfection,and samples were collected at 24h after virus infection.SYBR GreenⅠfluorescence quantitative PCR and TCID₅₀detection were performed.The results showed that the m RNA level of TGEV N gene and virus titer in the overexpression group were significantly lower than those in the control group,indicating that the overexpression of IFI44L inhibited the proliferation of TGEV.Three si RNAs targeting pig IFI44L gene（named si IFI44L-137,si IFI44L-419 and si IFI44L-1172 respectively）were further designed,and si IFI44L-137 was found to be the best by q RT-PCR.On this basis,si IFI44L-137 was transfected into PK-15 cells and then treated with virus infection 24h later.The samples were collected 24h after virus infection for q RT-PCR and TCID₅₀assay.The results showed that knocking down IFI44L promoted the proliferation of TGEV.These results indicated that swine IFI44L exibited a significant inhibitory effect on TGEV proliferation.3.Preparation of murine polyclonal antibody against porcine IFI44L proteinFirst,the prokaryotic expression recombinant plasmid p ET-28a-44L was constructed by inserting the IFI44L gene into the p ET-28a（+）vector.The recombinant protein was induced and purified,and BALB/C mice were immunized with the purified protein for 4times at an interval of 2 weeks.After the fourth immunization for 1 week,mouse polyclonal antibody（Pc Ab）against porcine IFI44L protein was prepared.Indirect ELISA showed that the antibody titer of the prepared mouse polyclonal antibody was 1:3 200.Western blot analysis showed that the Pc Ab could specifically recognize prokaryotic and eukaryotic expression of porcine IFI44L protein.Indirect immunofluorescence（IFA）assay showed that Pc Ab could react with porcine IFI44L in PK-15 cells.The IFA assay also revealed that porcine IFI44L was widely expressed in the cytoplasm and nucleus,but mostly in the latter.These results demonstrated the specificity and stability of mouse polyclonal antibody against porcine IFI44L protein.