Cloning Of Porcine LTβR Gene And Its Function On IPEC-J2 Cells

Enteric diseases caused death of piglets before weaning makes huge economic loss for the pig industry.Gut immune system which is composed by immune cells,immunoglobin A（Ig A）and intestinal microbes,play the important role in protecting piglet against infections.As one memeber of Tumor Necrosis Factor（TNF）super family,Lymphotoxinβreceptor（LTβR）has been reported to be critical for formation of lymphoid organs,and it plays the key role in immune response through regulation of the IgA secreation.It has been already shown that the LTβR^-/-mice are sensitive to bacterial infection due to the absence of lymphoid organs,and LTβR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1,and CXCL2 chemokines.We speculate that LTβR plays the important role in porcine gut immunity.To study the effects of LTβR genes on Porcine Small Intestinal Epithelial Cell Line（IPEC-J2）cells,further on gut immune responses in pigs,we cloned and charicterzied the porcine LTβR genes.The main findings and conclusions were listed as follows:1.The porcine Lymphotoxinβreceptor（LTβR）gene was cloned by RT-PCR method,the coding sequence（CDS）of porcine LTβR 1515 bp（predicted molecular weight is 45.58 kDa;pI is 5.78）and encoded 421 amino acids.The sequence homology analysis revealed that the S.scrofa LTβR had 81%and 78%identity with Mus musculus,and Homo sapiens respectively.2.The expression profiles of LTβR were examined by Q-PCR method.Our data showed that LTβR is highly expressed in liver,spleen,lung,kidney and adipose tissues,while the extreme low expression level was observed in muscle and heart.We also detected its expression in different gut sections and found that it highly expressed in jejunum while the lowest expression was observed in duodenum.3.Different dose of Lipopolysaccharide（LPS）and Interferon gamma（IFNγ）were used to treat IPEC-J2cells for 14 hours and the unchanged of expression of LTβR demonstrated that this gene can’t be induced and it mediated pathway can’t be activated by LPS and IFNγ.4.Furthermore,the genomic sequence spans 5’UTR to exon 3 was obtained by PCR.And the A-G mutation was identified in intron 2.The allele frequencies were examined with different pig breeds,including Duroc,Large White,Yorkshire and Chinese endogenous pig breed,Min pig.Results showed that the genotype frequencies of the loci were significant different among the examined populations.5.By Crisper/Cas9 technique,LTβR^-/-IPEC-J2 cells was generated,and the biallelic rate is 9.38%.In detail,the total of 96 single clones were screened and 22 of them were monoallelic mutants and 9 were biallelic mutants,all of 9 biallelic mutation clones were further confirmed by sequencing analysis.6.The knockout efficiency of Clone 1-10#was confirmed at both mRNA level（Q-PCR）and protein level（Western blotting）,results showed that LTβR gene expression level was significantly decreased.Further,we examined the expression levels of three LTΒR downstream genes,including VCAM1,IL22and IL23,in both wild type cells and in LTβR^-/-IPEC-J2 cells by semi-Q-PCR.Our data showed that both VCAM1 and IL22 were significantly decreased in LTβR^-/-cells.We then used the clone10 for the further studies.7.We also detected the effect of LTβR on IPEC-J2 cell prolerfation by CCK assays.Our data showed that ablation of LTβR inhibited IPEC-J2 cell proliferation ability.Semi-PCR reported that PCNA proliferating marker was reduced in LTβR^-/-cells.To understand the potential mechanism whereby LTβR affect IPEC-J2 cell proliferation,we performed the flow cytometry using propidium iodide to analysis the cell cycles of LTβR^+/+and LTβR^-/-.Our data demostrated an S phase arrest in LTβR^-/-cells,which resulted in the siginificant increase of S phase population.This result make us speculated that knockout LTβR don’t allow cells to pass the S phase and move to next cell cycle activity,which leads to the cell growth inhibition.8.Finally,apoptosis assay was carried out by HoloMonitor M4 Microscopy.M4 microscopy results clearly demonstrated that the knockout LTβR significantly induced cell apoptosis.The expression levels of apoptosis realted genes were detected by QPCR in both LTβR^+/+and LTβR^-/-cells.Several key apoptotic related genes,such as p53,BCL2,TRAIL-TNFSF10 and CASP3 were significantly up-regulated in LTβR^-/-cells.In conclusion,we cloned porcine LTβR gene and profiled the expression levels in different tisses.The LTβR gene null IPEC-J2 cells were generated by CRISPR/Cas9 technique successfully and knockout of LTβR could promote the cell proliferation and apoptosis.Our data is a first step for further understanding the biological role of porcine LTβR gene and to investigate whether it can serve the marker for pig immunological traits in pigs.