Biopanning The Interaction Protein With Host Intestinal Epithelial Cells From Trichinella Spiralis

Trichinellosis is a global zoonotic parasitic disease caused by nematode. It is stillprevalence in China. More than 150 mammals could be infected besides human.Trichinellosis could not only cost great expenses in animal industry but also threatenpeople's health. After the new host ingest the meat or meat food products whichcontain T.s infective L1, the larvae were released when the muscle tissue and cystswere digest by pepsin in the stomach. This process required a few minutes, it was notup to one hour. When the larvae were released, they invaded to the villi of smallintestine immediately and passed into the cell niche that composed of 117 cellulacolumnoepithelialis. The larvae were assigned a name T.s infective L1 at intestinalstage after this process. Strangely, the columnar epithelium cells don't split, butexpand, and the cell membrane fusion become niche to protect the larvae. But themechanism of that is not known now.It has been speculated that the invade process were probably caused by somekind of protein secretion or cytokines which could be secreted by T.s. The factorsregulate epithelial cells changed or fusion, so that they could accommodate T.sinfective L1 at intestinal stage. If we can obtain the specific genes during this periodof Trichinella, we will reduce the binding capacity between T.s infective L1 atintestinal stage and intestinal columnar epithelium cells, and enhance the capacity ofintestinal worms emission. That will bring a broad prospects about prevention ofTrichinellosis. From the existing biotechnology, it is an effective method thatbiopanning the interaction protein gene by phage display technology.The phage display technique takes the phage as a carrier, made protein or themultipeptidegenes inserted into outer covering protein gene areas of phagesdirectionally, which caused the extraneous proteins or the multipeptidesthroughexpressed fusion proteins with the phage outer covering proteins and displayed on the surface of the phages. The proteins or multipeptideswhich were displayed mightmaintain the relative independent spatial structures and biological activities. And thenbiopanning the expression special protein or multipeptidephages used affinityenrichment. The T7 phage display system was the newest one which was developedby Novagen Corporation. It could display broadspectrumproteins or multipeptides.The expressed multipeptidesor proteins as fusion proteins which were displayed onthe surface of the phages with package of membrane proteins of phage.Construction T7 phage display library of T.s infective L1 at intestinal stageInfective larvae at intestinal stage of Trichinella spiralis are collected afterinfected wistar mouse. Total RNA was extracted by Trizol reagents? Messenger RNAwas isolated from total RNA by Oligotex Centrifugalization? Then mRNA wasreverse transcribed into doublestrandedcDNA, end modification, EcoRI/HindIIIadaptors ligation, the dscDNA was disgested with HindIII and EcoRI. Afterfractionated by Mini Column, the dscDNA could be ligated into the T7 Select 103bvector arms. After packaged in vitro and amplificated, the quality of the library wasidentited. The result was the primitive library capacity was 6.0×10 5 Pfu, and the title ofamplification library was 1.9×10 12 Pfu/mL, recombinant ratio was 98%, the ranges of95% inserts were 2502000bp.Small intestinal epithelial cell isolation and primary culture of newborn pigsSmall intestine was removed from abdomen in aseptic conditions and then wascut into many fragments that was 1520cmevery fragment. Poured into collagenasedigestive juice which preheated at 37℃, both ends sutured and washed with DHankssolution, digested 30min at 37℃. Collected intestinal contents and centrifuged at 400gfor 7min. Cell sedimentum was resuspendedwith DMEM culture medium whichcontains 10% FBS. Washed twice, and the dispersed IECs (Intestinal Epithelial Cells)were plated on cell plates in DF medium at 37℃in a 5% CO2, after 48h culture, IECswere passaged until they could be fusion or be able to subculture. The IECs thatisolated by this method had good morphology, were short spindle or polygonal, andsome like pebbles. The results showed that IECs were forming cell colony in 46dand growing the full of plates in 1012d.The shape of IECs were polygon and thebouncary of cells was distinct. It was showed that 85% IECs were positive with HEstaining and immunohistochemical identification.Biopanning the interaction protein with host intestinal epithelial cells fromT.s infective L1 at intestinal stageIncubated the amplified phage display library with IECs 4 hours at 37℃, mixedthem with organic phase which was composed of dibutyl phthalate: cyclohexane as9:1 (v:v), put them into liquid nitrogen 3 min after centrifugation. Eluted phageswhich combined with IECs with phage extraction buffer, went on next biopanningafter amplified the phages. Picked up the positive phages and PCR amplified, detectedthe fragment size by electrophoresis and sequenced by BGI. There were four geneswhich were separately named IECTsp1,2,3,4. IECTsp1,2,3 were known geneof Trichinella sipralis, but IECTsp4was unknown gene. The 2D molecularconformation displayed that it was composed of many alpha helix, random coil and alittle extended srtand.