Immunoscreening Of Trichinella Pseudospiralis (NBL) CDNA Library And Sequence Analysis

Trichinosis, also called trichiniasis or trichinellosis, is a food-borne diseases,mainly because of the ingestion of raw or semi-cooked meat with parasite larvae. Thedistribution of this disease is extremely extensive, concentrated in the developingcountries in Southeast Asia and some developed countries with the habits of hunting.The reason why the reports of the disease has greatly increased in recent years may bethat with the improvement of living standards, the demand on the meat food has alsoincreased which led the spread of the distribution of the disease, meanwhile thecrossing-regional tourism also is also an important reason. In the countries of NorthAmerican, European and Australia, the consumption of beef, horse and bear meatinfected with trichinella spiralis and other meat related products also contributed to animportant cause of human trichinosis. In the provinces of Guangxi, Tibet and Yunnanof China, the local eating habits lead to the incidence reported, which caused thesocial impact of the extremely serious.In the reported12trichinella species, trichinella pseudospiralis is the onlyone that can infect both mammals and birds kinds which is different from others,also. Because of its pecial characteristics it’s difficult to prevent and to control. Adultworm of trichinella pseudospiralis could last for30days in host intestinal, and thenew born larva has4month to infect host muscle’s whole cell, which made the patienta long time pain. The diagnosis way of this disease is direct muscle compressor or tocheck up after muscle digestion with the light microscope mainly, because the cysticaof trichinella pseudospiralis in the muscle is very thin, so the traditional way is useless,meanwhile it is impossible to examine human muscle out of moral reasoning. Surfaceantigens and excretion-secretion antigens of different period of trichinellapseudospiralis are different, but all could induce the host immune response. Reportsshows that the new larvae is stage that only transmigrate through blood but don’tgrowth phase. And the host immune to is very sensitive, so this stage of worm is important for the immune system regulating of host. The survival time of Musclelarvae is up to several years or more in host muscle cells, antibodies produced thisperiod could be used for diagnose. If the antibody can also recognize the antigens ofthe newborn larva and adults, it will be a way to identify specific genes of the parasitethroughout the life cycle.This experiment used trichinella pseudospiralis （ISS13） newlarvae cDNA library is complete with experimental standards after testing the titerwhich is4.25x107pfu/mL. Before the immune screenin of the cDNA library,we mustfirst amplify nonspecific blue phage make sure the titer of it is up to109pfu/mL. Andthen to get rid of the nonspecific antibody of E.coli protein and blue phage proteinusing the method of “false screening”,in order to ensure the effectiveness andspecificity of the immune screening on trichinella pseudospiralis newbornlarvae cDNA library. After the optimization of the screening conditions using themethod of ELISA, the specific antiserum is on a good condition for the cDNAlibrary screening of trichinella pseudospiralis newborn larvae after the first round ofscreening,54positive clones were obtained. Then a second screening was done basedon the first positive clones, finally10positive clones were obtained. The last step is tosequence the finally10positive clones and to process the sequence analysis to thefurther biological features. The10results of sequenced genes show that1was knowngene, named HW7,with a100%homology of ABL09499.2;2unknown genes, namedHW1，HW9.The other7genes were all new genes, named HW1、HW2、HW4、HW5、HW6、HW8、HW10respectively.HW1owns the homology of84%with the gene ofXP₀03378739.1[Trichinella spiralis], which coding the protein of gliomapathogeneis-related protein1;HW2owns the homology of36%with the geneofXP₀03378739.1, which coding the protein of conserved hypothetical protein;HW4owns the homology of82%with the gene of NP₀77265.1[Trichinella spiralis],which coding the protein of cytochrome c oxidase subunit I;HW5owns the homologyof81%with the gene of XP₀03373067.1[Trichinella spiralis], which coding theprotein of deoxyribonuclease II superfamily; HW6owns the homology of96%withthe gene of XP₀03373863.1[Trichinella spiralis], which coding the protein ofprohibitin; HW8owns the homology of93%with the gene of XP₀03370272.1[Trichinella spiralis], which coding the protein of calcium-activated potassium channel slo-1；HW10owns the homology of92%with the gene of XP₀03380590.1[Trichinella spiralis], which coding the protein of neuronal calcium sensor1. In thepositive clones, HW5was found stage specific, which could code the protein having ahigh homology with DNase II gene of Trichinella spiralis. DNase II could cause thegene transcription of and expression abnormally, having something to do with theform of the thin collagen cyst.At present the researchers of our laboratory have completed immunoscreening oftrichinella pseudospiralis newborn larva cDNA library using the anti-serum obtained26days and60days after oral infected. In this paper, we successfully use32days ofanti-serum after infected to immunoscreening the newborn larvae period of cDNAlibrary, and finally obtained related gene effects. Based on this research, we couldexpress the screened genes to establish a sensitive, specific diagnose method, as wellas lay the foundation for the trichinosis early diagnosis and vaccine research even forthe furhter study of gene function research.