Cloning And Expression Of AcGal Of Angiostrongylus Cantonensis, And Analysis On The Immunoreactivity Of The Recombinant Antigen

Angiostrongyliasis is caused by Angiostrongylus cantonensis. As a kind ofnewly emerged endemic disease through eating, the epidemic situation ofAngiosrongyliasis is becoming more and more serious all over the world, and isshedding more and more threats on human beings. In human, A. cantonensis mainlyinvades the central nervous system and causes eosinophilic meningitis ormeningoencephalitis. The most common symptoms and signs are headache, nausea,vomiting, fever, neck stiffness, paraesthesia, and weakness of extremities, muscletwitching, diplopia, strabismus, facial paralysis and somnolence. Among them,headache is most serious and typical signs. Some patients explained that theirheadaches were so severe that they could not suffer from it. Muscle pain andparesthesias are also serious. Some patients felt heavy pain even when they changedtheir clothes and the pain usually could not be relieved with antalgica. Symptoms likepsychosis were also found in few patients.China is one of the main endemic countries. There were growing incidences ofangiostrongyliasis recent years in China. An epidemic of this disease even broke outin some area. It is urgent and important for us to control this disease efficiently atpresent. Diagnosis of Angiostrongyliasis is the key point in the control work. There isstill no sensitive, specific and mass producible standard diagnosis kit in China up tonow. To solve these problems, the following researches were carried out.1. Finding and primary research of the AcGal gene from cDNA library ofAngiostrongylus cantonensisdThe full-length ORFs of AcGal gene was amplified from the A.cantonentsislarvae cDNA library. The gene function of the gene was analyzed by homologysearch through BLASTX program and CD-search (Conserved-Domain search).AcGal was highly homologous to Hypothetical Galectin protein 10 fromCaenorhabditis elegans, This domain exclusively binds beta-galactosides, such aslactose, and does not require metal ions for activity. GLECT domains occur ashomodimers or tandemly repeated domains. They are developmentally regulated andmay be involved in differentiation, cell-cell interaction and cellular regulation.2. Cloning and expression of the AcGal gene of Angiostrongylus cantonensisdThe AcGal cDNA sequence was amplified through PCR. Then, the amplifiedfragment was subcloned into the expressional vector pET32a(+) and was expressed inE. coli.. The inclusion body was washed, degenerated, refolded by dialyzed, thencondensed. Furthermore the protein was analyzed by SDS-PAGE and Western-blot.The recombinant protein of AcGal expressed with pET32a(+)/BL21 system canreact with the infected rat's serum. The AcGal protein is supposed to be a potentialdiagnosis antigen.In conclusion, the AcGal gene of Angiostrongylus cantonensisd was firstlycloned and identified, the recombinant AcGal may contribute to further study in thediagnosis of Angiostrongyliasis.