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Objective To construct cDNA library from adults of Anopheles dirus for cloning the immune genes or related genes for malaria parasites development. Methods The mRNA of adult Anopheles dirus was isolated. The library was constructed by using the Zap Express vector(Stratagene) and the quality was evaluated. Results The efficiency of the library was 2.1?10 6 pfu/ml with 98% clones positive. The average length of the insert fragment was over 1 kb. Conclusion cDNA library of adult Anopheles dirus with high efficiency can be constructed by using the Zap Express library construction Kit (Stratagene).
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Objective To explore primarily the differentially expressed proteins in the hemolymph from adult female Anopheles stephensi ( An stephensi ) infected with Plasmodium yoelii ( P yoelii ) after being fed with sucrose solution containing nitroquine or not at different time points Methods Hemolymph of 2 groups of adult female An stephensi was collected with the expulsion method from the first day to the fifth day after the feeding Hemolymph samples were examined with SDS PAGE The protein gels were visualized by either Coomassie brilliant blue or silver staining, scanned and automatically analyzed by the BioRad1000 gel image analysis system for differential proteins bands Results On the second day of feeding with nitroquine, a few oocysts were partially melanized Furthermore, during the period from the fifth day to the ninth day, the number of mosquitoes with malanized oocysts and the number of melanized oocysts gradually increased The number of hemolymph protein binds in the treatment group was markedly more than that in the control Many different bands, mainly located at the molecular weight of (20～40)?10 3 and (60～80)?10 3, were visualized in the 2 groups The number of protein bands stained by the silver staining was more than that by the Coomassie brilliant blue staining Conclusion There are differentially expressed proteins in the hemolymph in An stephensi infected with P yoelii after being fed with sucrose solution containing nitroquine These differential proteins may be the melanization engaging proteins
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Objective To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.
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Objective] To analyse the soluble antigens of different developmental stages of Pagumogonimus skrjabini and deve lop a specific and sensitive serodiagnostic method for pagumogonimiasis. [Methods] The soluble antigens of P.skrjabini of various stages were separated by SDS PAGE. The specific antigen of the adult fluke was recognized immunologically by immunoblot assay. The protein bands between 10～30 kDa purified by SDS PAGE and electrophoretic elution were used in dot ELISA. [Results] Using dot ELISA, the soluble antigens of adult were recognized by sera infected with P skrjabini . More reactive bands appeared at 10～30 kDa, but major protein bands were at 22、24 and 26 kDa. However, using sera from patients infected with other trematodes including schistosome and Clonorchis , cross reaction bands appeared within 60 to 90 kDa. When compared with ELISA of crude adult antigens for detecting 28 suspected patients, there was no significant difference between the two methods. The sera of 38 patients with other diseases were also detected by the two tests. No cross reaction occurred with the purified adult antigen dot ELISA while 13 2%(5/38) of the sera cross reacted in ELISA of crude adult antigens. [Conclusion] Dot ELISA using 10～30 kDa antigen might be a specific and sensitive serodiagnostic method for diagnoing pagumogonimiasis.
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AIM:To cultivate the exoerythrocytic stage of Plasmodium yoelii in vitro and to study some involved affecting factors.METHOD:In vitro cultivation.RESULTS:The monolayer hepatocytes grown in 1 6 - mm plastic cell culture dishes were inoculated with sporozoite sus- pension prepared from Anopheles stephensi mosquitoes for48hours.At a final density of2? 1 0 4 cells per well,the infection rate of hepatocyte,cultured in medium supplemented wit15 % bovine serum,was 0 .0 35? 0 .0 1 3% ,the diameter of the nearly mature EEF of Plas- modium yoelii was up to40 .3? 31 .6 ?m,and contained more than1 0 0 nuclei,the number of EEF might be4- 1 0 /cm2 .An intraperitoneal inoculation of the EE schizonts to mice could induce parasitemia.At a final density of0 .5? 1 0 4 or4? 1 0 4 cells per well or the hepatocytes cultured in medium supplemented with1 0 % bovine serum,no EEF could be observed.CON- CL USION:The density of hepatocytes and culture medium are important for the cultivation of the EE stage of Plasmodium yoelii.This procedure will lay foundation for the further studies of the sporozoite invasion,the development of EEF and the affecting factors in- volved.
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Objective To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. Methods The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Plutonic F-127 under confocal laser scanning microscope (CLSM) at different time. Results The best load condition was that the oocysts were incubated in 3 ?mol/ml Fluo-3/AM adding 1 ?l/ml 25% Pluronic F-127 for 60 min at 37 ℃ . Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2 + in the mature oocysts was (137.15 ?7.02) nmol/L(X?S) but was (18.44? 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. Conclusion The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-reiractory anopheline mosquito species.
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Objective To analyze the protein in hemolymph from adult female Anopheles stephensi (An. Stepheni) infected by Plasmodium yoelii after feeding with sucrose solution containing nitroquine or simple sucrose solution with two dimensional gel electrophoresis technique. Methods Hemolymphs from nitroquine fed, infected blood fed, and sucrose solution fed adult female An. stephensi were collected using the expulsion method on the third day after the feeding. Hemolymph protein concentration was examined with Bradford method. Then the hemolymph protein was analyzed by two dimensional electrophoresis. The protein spots were visualized by Coomassie brilliant blue staining. The spots were scanned and automatically analyzed by the ImageMaster VDS CL (Amersham Pharmacia) and ImageMaster 2D Elite software (Amersham Pharmacia). Results The protein concentration in the nitroquine fed group was always lower than that in the infected blood fed and sucrose solution fed groups. Two dimensional gel electrophoresis revealed 101 protein spots in nitroquine fed and 115 protein spots in the control with 51 matched, but unmatched 50 and 64 protein spots were detected in the treatment group and the control group, respectively. Different protein spots were mainly located at the molecular weight of (40-60)?10 3 and at the isoelectric points of basic end. Conclusion Two dimensional gel electrophoresis may directly reflect the difference of the protein. Both the difference of protein concentration and the protein spots may be involved in nitroquine induced melanotic encapsulation of Plasmodium yoelii oocysts.