Cloning Of Prophenoloxidase Gene Of Anopheles Dirus And Study On The Immune Defence Reaction Against Plasmodium Yoelii Infection

Malaria, transmitted by a mosquito vector,is a severe infectious disease with high incidence rate and fatality rate.Control of malaria will be confronted with great challenge because of the rapid spread of multi-drug resistant parasites,the emergence of insecticide-resistant anopheline mosquitoes and the lack of effective vaccines fighting against malaria,which have emphasized the need for new effective malaria control strategies. 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 that support or impact the development of malaria parasites in the mosquito vecter and the study on the mechanisms of innative immune defence responses against Plasmodium infection.The melanotic encapsulation of oocysts is a specific immune defence mechanism against Plasmodium infection ,and is also a primary phenotype in the Plasmodium-refractory strain of An.gambiae. Prophenoloxidase(PPO) is a key enzyme for mediating melanotic encapsulation of oocysts,which may be transformed into active phenoloxidase(PO) by limiting proteolysis. The active PO can catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine and the oxidation of dihydroxyphenylalanine and dopamine to their respective quinones.The reactive quinones serve as a defence response by mediating protein cross-linking and polymerizing to form a melamin layer that immobilizes the pathogen, and they are also thought to be toxic to the invading organisms.The melanotic phenomena mediated by PPO is widespread in both mosquitoes and other insects that being involved in sclerotization and canning of the egg shell and cuticle,wound healing and encapsulation of invading microorganisms.The multiple functions of melanization cascades are reflected in a large number of PPO genes present in mosqito genomes.Up to date, six PPO genes in An.gambiae have been identified,but only one PPO gene from An.stephensi or An.culicifacies has been cloned,respectively.Preliminary investigations have been made on the role of PPOs of Anopheles mosquito against Plasmodium infection. An.dirus is the main mosqito 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 midgut.Now,in spite of many correlated studies on An.dirus for transmitting P.falciparum,there is less investigations on the mechanisms of innative immune defence against Plasmodium infection, especially on the immune defence response against P.yoelii infection.Based on the above reasons,several basic researches will be made.Firstly,complemental deoxyribonucleic acid (cDNA) of PPO gene in An.dirus will be cloned. Secondly,affect of blood-feeding and infection of P.yoelii on the transcriptions of PPO gene of An.dirus will be assessed.Thirdly,the distributions and the changes of PPO in the midgut of An.stephensi and An.dirus at pre-and-post-infection with P.yoelii will also be investigated.The experimental contents and main results are shown as follows:1. Degenerate primers were designed according to the conserved amino acid sequence of PPO from nine insects,such as An.gambiae,Manduca Sexta ,et al.RNA sequence of the larva of An.dirus was amplified by RT-PCR to get 600bp PPO cDNA segment.The products of RT-PCR were cloned into pMD18-T vector after being purified,and transformed to XL1-Blue strain.70 white colony approximately were harvested in the plate of LB culture with aminobenzylpenicillin.A single white colony being sought out at random was cultured for extracting their plasmids.The plasmids of TA clone were hydrolyzed by Ecor I and Hind â…¢ and got a band of DNA beyond 600bp.Furthermore,the positive plasmids confirmed by enzyme digesting were dilluted and amplified by PCR and got a band of DNA about 600bp that was the same as the band amplified usin...