Diagnosis, Screening And Expression Evaluation Of Parasitic Insect Pests

Human pentastomiasis is an unusual parasitic zoonosis, which is usually caused by wormlike pentastomida or "tongue worm", and mainly located in countries or regions in Africa and Southeast Asia etc. People get infected by eating pentastomida eggs or the animal innards containing infective larvae. In China, the cases of pentastomiasis are mainly reported in Zhejiang, Guangzhou, Shandong, Taiwan Provinces, etc, and the main pathogenic species are L.serrata, A.agkistrodontis, A.moniliformis and P.taiwana. Up to now, the methods for diagnosing pentastomiasis are comprehensive diagnosis based on the morphology, histopathological and imageological examinations combined with clinical manifestations and epidemiology. However, the relative serological and molecular tests are rarely reported, which leads to many clinical misdiagnosis and missed diagnosis. Hence, it is highly required to develop fast and effective in-vitro diagnostic reagents for pentastomiasis. It is known that, the diagnostic antigen is the key to develop diagnostic reagents and the recombinant antigen is one of the important sources of diagnostic reagents. In this thesis, with the aim of obtaining the valuable diagnostic antigens and using A.agkistrodontis nymphs as the research materials, we complete the following researches:the study of crude antigens of A.agkistrodontis nymphs, the construction and immunoscreening of cDNA library and the expression and evaluation of recombinant proteins.In our research, A.agkistrodontis nymph was used for experiment. Firstly, the crude antigen of A.agkistrodontis nymphs was obtained by repeated freeze-thaw procedure; The components and immunological properties of the crude antigen, as well as the diagnostic effects of the crude antigen were observed and analyzed. Meanwhile, the constructions of cDNA entry library and cDNA expression library are implemented based on Gateway Technology, and the cDNA expression library was immunoscreened using the mixed sera of mice infected with A. agkistrodontis. Then, the homology of the screened genes and their encoded proteins were searched in Genbank, and the structures and functions of predictive encoded proteins were also analyzed by bioinformatics softwares. Finally, the positive clones were expressed in prokaryotic cells, and the recombinant proteins were purified, as well as the diagnostic effects of the recombinant proteins for detecting the sera of mice infected with A. agkistrodontis were evaluated.The results showed that, the crude antigens of nymphs were separated into9main-bands and13sub-bands （Mr16-150kDa） in SDS-PAGE gel. Among them,16and5immune reaction bands were recognized by the mixed sera of mice and patients with pentastomiasis respectively on Western Blot. Besides, slightly cross-reactions with sera of paragonimiasis and trichuriasis were observed at about Mr34kDa and no cross-reaction was founded with sera of other parasite diseases. The sensitivity and specificity were all100%using the crude antigens as a coating antigen to detect sera of patients, mice with pentastomiasis and normal people, and no cross-reaction was observed with sera of patients with other parasitic diseases,.The cDNA entry library and cDNA expression library of A.agkistrodontis nymphs were first successfully constructed. The average titer and total clones of the cDNA entry library were1.45×105cfu/ml and1.74×106cfu, respectively, and the range of fragment length of the inserted cDNA was between0.2-4.0kb, with an average of1.4kb. The range of fragment length of the inserted cDNA in the cDNA expression library was between0.3-2.2kb, containing a total clones of105cfu, with an average of1.0kb. Twenty-one of positive clones were obtained by primarily screening the cDNA expression library using the sera of infective mice with A. agkistrodontis, seven of them, coded S1, S5, A1, D1, F1, P1and P5clone, respectively, were analyzed. The sequence and homology of seven positive clones were sequenced and analyzed by the blastn program. No significant similarity was founded in pentastomida species, which meant that they were all the novel genes of A. agkistrodontis. However, among these genes, Dl gene had a higher homology with USDA-FP₁85181gene of77%from Lysiphlebus testaceipes. The blastp search showed that the homology of the seven predictive proteins S1, S5, Al, Dl, F1, P1and P5were similar to AGAP004551-PA of Anopheles gambiae str. PEST, hypothetic protein of Amblyomma maculatum, collagen alpha-1, IV, chain precursor of Pediculus humanus corporis,40S ribosomal protein S5a-like of Bombus impatiens, CG8092CG8092-PA of Tribolium castaneum, putative protein kinase C inhibitor of Hottentotta judaicus, ribosomal protein L19e of Tribolium castaneum, with identities of27%,45%,56%,86%,30%,61%and86%, respectively. Then, we chose four of seven positive clones （A1, D1, P1and P5） for the next recombinant expression and purification, and the sensitivity of detecting the sera of mice using the four recombinant proteins was100%,100%,85%,100%, respectively,while the specificity was95%,90%,100%and95%, respectively.In summary, the crude antigens of A.agkistrodontis nymphs can give a better diagnostic effect in detecting the pentastomiasis by indirect ELISA. The cDNA entry library and expression library constructed by Gateway technology provide rich materials for genetics research of pentastomida and form a foundation for further studying on the gene functions and screening for the immunodiagnostic antigens. Seven positive clones screened are all the new genes of A.agkistrodontis, among which, A1,D1, P1and P5recombinant proteins are expressed in the prokaryotic cells, purified and evaluated. Except PI, the diagonistic sensitivity and specificity of A1、D1、P5recombinant proteins are similar to the crude antigens. However, further study is required on whether these recombinant proteins can be used for the detection of human pentastomiasis.