The Construction & Immunoscreening Of CDNA Expression Library Of Mucscle Larvae Of Trichinella Spiralis & Trichinella Pseudospiralis

Trichinellosis was a global zoonotic parasitic disease caused by nematode. It was still prevalence in China. More than 150 mammals could be infected besides human.Trichinellosis could not only cost great expenses in animal industry but also endanger to human health . Trichinella spiralis muscle larvae is infected with host, experience four exuviations to develop into imago in 30h.Consequencely,it gave birth to newborn larvae in 96h and newborn larvae invaded into all skeletal muscle by lymph and blood. Finally, newborn larvae grew up infective muscle larvae, which could survival in animal and human body for long term. Muscle larvae take in host nutrition and endangered to host health. The multiplicity Trichinella spiralis antigens made a difficulty for a large-scale reproducing in vitro,immunodiagnosis and prevention .Recently, Trichinella spiralis study only focus on antigen, but its antigen is multiplicity. Though ES was used of immunodetection in trichiniasis ideally, however, epitope of ES had nonspecific reaction with factor of human serum. Meanwhile, it could lead to a false positive diagnosis and there were difficulty for large- scale preparation. In this study the muscle larvae (ML) cDNA library ofTrichinella spiralis and T.pseuodospiralis was successfully constructed of with lambda ZAP Express vector at first,then the ML cDNA library of Trichinella spiralis and T.pseuodospiralis was immunoscreened and antigen genes were cloned with the molecular biology and immunologic technology. This will lay the foundation to obtain diagnostic and protective antigen of Trichinellosis.At first, in order to clone and study functional genes of muscle larvae of Henan Trichinella segregation of pig in china Trichinella spiralis ( T.s)( International numbers:ISS534), cDNA library of muscle larvae of T.s was successfully constructed of with lambda ZAP Express vector. Total RNA of muscle larvae of Trichinella spiralis were extracted by the Trizol reagent. The original library contained 1.92×106 cDNA clones, the titer of amplified library reached 1.6×109, in which about 98.6% clones were recombinants and most of insert DNA fragments were between 0.4~2.0 Kb. cDNA library of muscle larvae of Trichinella spiralis was screened using the infected swine serum for 60 days with Trichinella spiralis and positive clones were sequenced and analysed. from 2. 5×105 recombinant clones15 positive clones were obtained. Sequence analysis show that 10 of them are the same gene, encoded protein is 99% homology to serine protease inhibitor of [Serine protease inhibitor] (AAF63473), nuclear sequence of WM5 is the longest and including ORF among the ten clones, in GenBank database WM5 is NO. ABI32311,there were mutations of three nucleotides in the ORF of the Chinese this cDNA comparing with that from Japan isolate at the positions 542,696 and 989, namely C,G and T in the Japan isolate but G,A and A in the Chinese isolate. , the mutation of the three nucleotides was considered as the single nucleotide polymorphic (SNP)marker of the Chinese Ts isolate, exclusion. mutations of sequencing WM20 without ORF showed high homology to Mitochondrion genome of Trichinella spiralis and encoded large subunit ribosomal RNA. The other 4 clones were never reported new genes. To obtain one stronger reactive antigenicity and high copy gene named WM5 which will probably become a candidate gene for the Trichinellosis diagnosis antigen. These results play a foundation part in the further study of the genic recombinant antigen of Trichinella spiralis.It is important to penetration of species and export and import of flesh。The second, in order to clone and study functional genes of muscle larvae of T.pseuodospiralis , cDNA library of muscle larvae of T.pseuodospiralis was successfully constructed of with lambda ZAP Express vector. Total RNA of muscle larvae of T.pseuodospiralis were extracted by the Trizol reagent. The original library contained 4.0×106 cDNA clones, the titer of amplified library reached 1.5×109, in which about 95.4% clones were recombinants and most of insert DNA fragments were between 0.4~2.0 Kb. cDNA library of muscle larvae of T.pseuodospiralis was screened using the infected swine serum for 60 days with Trichinella spiralis and positive clones were sequenced and analysed. from 2.0×105 recombinant clones 27 positive clones were obtained. Sequence analysis show that 11 of them are the same gene and now not still reported, encoded protein is proteasome activator subunit, nuclear sequence of RW29 is the longest and including ORF, Accession number is EF043393 in GenBank database; sequence of 8 clones is 100% homology to Tp21kDa mRNA; sequence of 3 clones is 98.7 % homology to Tp28kDa mRNA and Accession number of full RW26 in three clones is EF043393;RW44 and RW45 are the same unknown genes; the other three (RW53,RW23, RW19 ) are new genes and separatelly encoded differently prorein. The stronger reactive signal and high copy cDNA encoded proteasome activator subunit will probably become a candidate gene for the T.pseuodospiralis diagnosis antigen. These results play a foundation part in the further study of the genic recombinant antigen of T.pseuodospiralis.In this study,it promoted antigen of Trichinella spiralis muscle larva to become candidate gene, antigen gene recombination in Trichinella spiralis every growth stage, expression of polygenic recombination antigen and make foundation for establishment of sensitive and specific immunology detect method.