| Malaria,transmitted by mosquito vector,is a severe infectious diseases with high incidence and fatality rate in the world population. Over 3 billion people live under the threat of malaria. It kills over a million each year –mostly children (WHO:World Malaria Report 2005). Control of malaria will be confronted with great challenge because of the rapid spread of parasites with multiple anti-malaria drug resistance,the emergence of insecticide-resistant anopheline mosquitoes and the lack of effective vaccines fighting against malaria. Therefore,new concepts and means for malaria control are an urgent need. In the beginning of 1990s,WHO has suggested a new malaria control strategy by transforming the malaria-transmitting mosquito into harmless one. In order to reach this goal,the primary basic research work is to clone,evaluate and analyse the function of the genes which can support or impact the development of malaria parasites in the mosquito vector. The innate immunity of anopheles can limit the development of malaria parasites in the mosquitoes,which includes the activation of melanotic encapsulation,the production of NO or defensins. However,only the melanotic encapsulation can completely suppress the growth of malaria parasites and the other innate immune reaction only can adjust the infectious rate of parasites,the melanotic encapsulation is the most important immune reaction.The encapsulation may be initiated by binding of soluble pattern-recongnition receptors to the parasites surface factors and activation of a serine protease cascade that ultimately leads to proteolytic activation of prophenoloxidase. Active phenoloxidase then cross-links other protein and polymerizing to form a melamin layer that immobilizes the pathogen,and they are also thought to be toxic to the invading organisms. Up to date,different PPO,Sp and defensins in An.gambiae have been cloned,but how can the innate immunity be initiated is unknown. An.dirus is a main mosquito vector for transmitting malaria in the area of Southeast Asia and the southern mountain area of China, which is susceptible for P.falciparum and P.vivax,but refractory for P.yoelii owing to melanotic encapsulation of oocysts in the midguts. So An.dirus-P.yoelii is a suitable model for study of defense mechanism of mosquitoes. At present,the degenerate PCR and DD-PCR are used to obtain the related genes of immune mechanism of mosquito. However,the number of genes is so limited through these technology which cannot overcome the question of genes up-regulation. Therefore,in this study,based on the model of An.dirus-P.yoelii,we would use a new method-suppression subtractive hybridization(SSH) to construct subtractive cDNA library related to Anopheles dirus against Plasmodium yoelii. Moreover,through the preliminary identification and bioinformatics,we would predict the function of the differential expressed genes in the process of mosquito infected with parasites. Nevertheless,we investigated the transcription changes of T6 in pre-infection and post–infection of P.yoelii by RT-PCR and explored the relationship of this gene and innate immunity against parasites. The experimental contents and main results were shown as follows: 1. The method of suppression subtractive hybridization(SSH) was used to construct the subtractive cDNA library. Mosquitoes were classified into two groups,An.dirus infected with Plasmodium yoelii were used as tester (T) group,while uninfected An.dirus were used as driver (D) one. Through suppression subtractive hybridization,the differential expressed cDNA in the tester were then picked out and cloned into pMD18-T vector after PCR ampilification. Then the T vector was converted into XL1-Blue,and so a differentially expressed cDNA library of An.dirus could be constructed. The titer of the library was 2500 clones in 1ml conversion and 83% phages were recombinants. The average length of the insert was 200~600bp. So the properties of this library may be suitable for further study. 2. We had use the method of virtual northern blot,DNA sequencing and BLAST to analysis the differential expressed cDNA. The results showed that five cDNA were homologous to functionally known genes,three genes were homologous to An. gambiae EST database,and two genes were homologous to functionally unknown entries. According to the functionally known genes,the differential expressed cDNA might be related to innate immunity,capacity metabolism and singal transduction. 3. The DNASSIST software was used to predict and analyse the function of some gene related to innate immunity,including the content of GC,restriction enzyme mapping and the sequence of coding protein. The results revealed that there are conservative serine proteinaseamino acid sequence in the predicted coding sequence,which includes histidine,asparagic acid and serine. Because of the conservation of this amino acid sequence in the insect serine proteinases,T6 gene could be a serine proteinase definitely. 4. The transcription changes of T6 in pre-infection and post–infection of P.yoelii were investigated by RT-PCR. The results showed that the T6 gene in post-infection was largely transcripted than in pre-infection,and so this results showed that the increase tanscription of T6 may be correlation with infection of plasmodium. The high level of transcription of T6 might be available for melanotic encapsulation of P.yoelii oocysts because An.dirus can melanize and encapsulate the P.yoelii oocysts mediated by Sp. In conclusion, we had constructed the subtractive cDNA library by SSH and might isolated genes related to innate immunity,capacity metabolism and singal transduction. Furthermore,the prediction and analysis of one gene related to innate immunity by DNASSIST software could reveal that this gene may be Sp. And the analysis of RT-PCR could show that this gene may be related to the melanotic encapsulation. In a word, all these studies can lay a good foundation for further screening experiment,and can largestly facilitate the study of inmmune mechanism of mosquito.
|
PDF Full Text Request