Establishment of Pig (Sus scrofa) Small Intestinal Epithelial Cell Line with CD14 Gene Silencing and Its Effect Analysis on the Adhesion Ability to Escherichia coli
Abstract：Cluster of differentiation antigen 14 (CD14) plays an important role in both the innate and adaptive immune responses. This study aimed at establishing the short hairpin RNA (shRNA) interference vectors with pig (Sus scrofa) CD14 gene silencing to package them into lentivirus for transfecting pig small intestinal epithelial cells (IPEC-J2) and conducting function analysis on cell level to provide fundamental basis for function and action mechanism of CD14 gene. Based on the whole coding sequence of pig CD14 gene (GenBank No. EF626695.1), 4 interfere sequences were constructed which encoded shRNAs against pig CD14 gene and cloned into lentivirus expression vectors of pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3, pGLV3-CD14-4 and negative control pGLV3-CD14-NC. IPEC-J2 was infected by lentivirus solution after packaging successfully and qRT-PCR was used to detect the interference efficiency of CD14 gene. Lentivirus with the highest interference efficiency continually infected cells through medicinal sieve to obtain pig small intestinal epithelial cell line with stable CD14 gene silencing. Escherichia coli adhesion test was conducted to detect the adhesion ability change of E. coli F18ab and F18ac to IPEC-J2. The results showed that 4 shRNA vectors were constructed successfully and the packaged lentivirus could reduce the mRNA expression level of pig CD14 gene, in which pGLV3-CD14-3 had the best interference efficiency of 94.6%. The adhesion results showed that the E. coli F18ab's adhesion ability to IPEC-J2 significantly enhanced after CD14 gene silencing and E. coli F18ac did not. This result showed that CD14 gene silencing enhanced the susceptibility to E. coli F18ab in small intestinal epithelial cells, which illustrated that CD14 gene may play an important regulation role in which small intestinal epithelial cells resist infection by E. coli F18ab. The establishment of pig small intestinal epithelial cell line with CD14 gene silencing stably mediated by lentivirus offered important material for mechanism research of CD14 and toll-like receptors/interleukin-1?receptor (TLRs/IL-1R) signal pathway, which played important roles in intestinal immune response and pathogen defense caused by pathogenic microorganisms. This study successfully screened out the efficient shRNA vector which could exclusively interfere porcine CD14 gene expression, which also provides experimental basis for further studying the CD14 gene function and mechanism for the resistance to gram-negative bacteria infection in porcine intestinal tract at the cellular level.
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