Screening And Identification Of Antigenic Genes Of Trichinella Spiralis At Different Development Stages

Trichinella spp. has been found more than 170 years, unforturnetely, the artificial inspection methods for animal trichinellosis were still used in the slaughterhous, and also there is not early diagnostic method for human trichinellosis up to now, these problems became the main source to let the trichinellosis became the highest cost food born zoonostic parasitic disease in worldwide. At present, specific and sensitive immuno-methods have been widerly used for diagnosis of many pathogens and diseases, wherase it is not the case for Trichinellosis. The main problem is that Trichinella spp. has different development stages, in which the larva were with different antigens expression. In this situation, the antigens from one stage larva could not recognize the antibodies induced by the antigens from othe stages's larvae. For example, the excret-secreted (ES) antigen of muscle larvae (ML), easily obtained and widely used one at present, could not recognize the early antibodies induced by adult worm (Ad) and newborn larvae (NBL), in this case, a dangerous blind area of diagnosis was formed, in which the infection could not be detected before 19 days post infection (p.i.), wheras the NBL could developed to the infectious larvae after 14 days post infection (p.i.). To overcome the problem existed in antigens from ML, it is neccessory to use the antigens from the different developent stage's larvae as the diagnostic antigens, wherase concerning the dificulty for the antigens purification from Ad and NBL, and the life cycle of Trichinella could not be finished in vitro up to now, it seems impossible to get the sufficient and effective native antigens from different stage's larvae for practice diagnosis. Wherase following the development of molecular biology, it became possible to expess the effective antigen genes from different stage's larvae, and to use the recombinant antigens for the immuno-diagnosis.In this study, the expression cDNA libraries of Trichinella spiralis larvae at 4 different development stages including muscle larvae (ML) at 35 days p.i., intestine ML at 6 h p.i. (inML6), adult worm at 3 days p.i (Ad3). and 24 h old newborn larvae (NBL) were constructed and screened by the 26 and 60 days pig anti-Trichinella sera respectively. The subtracted cDNA libraries of Trichinella spiralis larvae at 3 different development stages including Ad3, adult worm at 5 days p.i. (Ad5) and NBL were constructed and screened by suppression subtractive hybridization (SSH). Total 11 antigenic genes were selected from 4 cDNA libraries, which include a serine protease gene WN18 from NBL, a serine protease inhabitor gene WM5 from ML, a cysteine protease inhibitors gene T626-55 from inML6 and 8 serine protease family genes from Ad3. Following stage-specific genes were selected from the subtracted cDNA libraries, which include a serine protease gene T668 (the same gene as WN18) from NBL, 15 Deoxyribonuclease II family genes from NBL, a zinc finger protein gene T54 from NBL, 12 Deoxyribonuclease II family genes from Ad3, a cuticle collagen gene T671 from Ad5. Except of 8 serine protease family genes from Ad3 reoresented by clone L23, 15 Deoxyribonuclease II family genes from NBL represented by clone T31D4-N5 and 12 Deoxyribonuclease II family genes from Ad3 represented by T3223-6, All of selected genes were expressed in prokaryotic expression system and the reactionogenicity of recombinant proteins were identified by western-blotting method using 26 an 60 days pig anti-trichinella sera respectively, in which 4 recombinant proteins of WN18, WM5, T626-55, L23 were confirmed with high reactionogenicity. The reactionogenicity level of these 4 recombinant proteins together with the recombinant proteins of P43, P53 and P45, three antigenic genes previously reported, were compared in one Western blot test with 26 an 60 days pig anti-trichinella sera respectively, finally 4 high abundance and high reactivity antigenic genes were confirmed, which were the genes of T626-55 from inML6, L23 from Ad3, WN18 from NBL, WM5 from ML. The findings of these antigenic genes lay the foundation for the immuno-diagnosis and inspection of human and animal trichinellosis based on their recombinant proteins. In the same time, it also provided the good candidates for preparation of protective agents for Trichinellosis.In the experiment, two variant DNase II family genes were surprisingly selected from the subtracted cDNA libraries of NBL and Ad3 respectively, in which NBL was with 15 members and 12 of it in Ad3, together with the unique one P43 previousely reported, total 28 variant DNase II genes were found in Trichinella spiralis. All members of it in NBL and Ad3 were in high homology, the alignment score of NBL members were with 60-99% in nucleotide and in amino acids, and with 83-99% in both for members from Ad3. Homology comparation among the members from NBL, Ad3 and also the unique one P43 from ML revealed that the alignment scors were only 16-30% amoon the members from the three development stages, which indicated the low homology of the DNase II from different stages.The characterization of DNase II from Trichinella were identified by expression of a full length DNase II cDNA term T31D4-N5 from NBL DNase II family. The result indicated that Trichinella DNase II revealed some unique characters and probably to be a new type DNase II comparing the 94 DNase II genes from all other available bio-organisms: 1) In gene phylogenetic tree of all the available DNase II sequences, All Trichinella DNase II was in a unique branch, and all of other DNase in another same branch; 2) Trichinella DNase II was localized outside of the larvae but not in the lysosome as usual; 3) It has the enzyme activity at pH 7.0 even pH 8.0, wherase normaly the DNase II has the activity between pH 4.5-6.5; 4) There is not the conserved histatine in the C-terminal of amino acid sequence, this amino acid has been predicted as the activity amino acid for all of the DNase II.1)The activity amino acid for Trichinella DNase II and also for all of other bio-organisms was predicted by the sequence alignments, a high conserved histatine were found in N-terminal of all available DNase IIs, and predicted as the activity amino acid site, it was the 123 histatine in N-termination of T31D4-N5 sequence (AAK16519) corresponding to the 130 histatine sit of human alfa DNase II sequence (NP001366). Site mutation was used to confirm this prediction, the renaturated fusion protein of T31D4-N5 lossed the enzyme activity after the 123 histatine at N-terminal of T31D4-N5 was replaced by Serine and Lysine, the result implicated that the predicted site was most probably the real activity amino acid for Trichinella DNase II and also for all of DNase IIs. The result was discrepancy with the prediction by Maclae (2003) that the activity amino acid was at C-terminal of DNase II, which is corresponding to the 295 histadine of human alfa DNase II sequence and 284 histatine of T31D4, in fact, this site in Trichinella DNase II sequences was not histatine, it was changed to serine or lysine.2)The transcriptions and expressions of the different stage's Trichinella DNase II were displayed by RT-PCR and immunochistochemistry, in which T3223-6 represented Ad3 family ,T31D4-N5 for NBL family and P43 from ML were tested. The result indicated that they are strictly transcripted and expressed stage-specificly. The T3223-6, it was only in adult worm and localized in epidermic cell under the cutile of worm; For T31D4-N5, it was in larvar from 5 day to 24 days old newborn larvae and localized on the same position as T3223-6; For P43 was transcriped and expressed in larvae from 17 days p.i. until as long as the parasite can stay and localized on the stickcytes and cuticle of larvae and in the cytoplasm of the nurse-cell;The potential function of Trichinella DNase II in the Trichinella invasion and survival were also discussed.1) There are some founctions involved in the cuticle formation, DNase II from AD can digest the DNA of epidermic cell and help it changed to cuticle. This procedure can avoid the release of parasite DNA to host and reduce the immuno-reaction of host when the molts of parasite were taken on.2) DNase II from NBL and ML as the endonuclease to cleave mitochondria DNA. The probably role of DNase II is that DNase II cleave mitochondria DNA to provides a hypoxia microenvironment, as the nurce cell become a low PH encironment after the mitochondria DNA be destroyed. Although DNase II have been suspected to act as a very important factor in DNA degradation in apoptosis and cell differential, we failed to detect the DNase II in the nucleus of nurse cells. It is possible that DNase II direct access to the cytoplasm in order to destroy the mitochondria DNA, which not only can provide a hypoxia microenvironment for the survive of T. spiralis, but also can avoid the mechanical damage because of muscle movement.3) DNase II as the endonuclease to downregulate the inflammatory reaction of host. We can image the scenario: The invasion of T. spiralis can causes the necrosis of a large amount of host cells, these necrostic cells released DNA into the circulation, which exceeded the local phagocytic clearance capacity. Finally, this undigested DNA cause strong immuno-reaction from host immune system, which will not favorite for the stay of the larvae in the host. The secretion of DNase II enzymes by T. spiralis may enhance the degradation of released DNA that not yet been cleared by phagocytes, which may reduce the host inflammatory reaction.