Construction Of CDNA Library From Adult Of Anopheles Dirus, Cloning Of Pro-phenoloxidases And Study On The Effect Of Malaria Parasite Infection On The Expression Of Them

Malaria is a severe infectious disease with great damage to the health, economic and social development of the epidemic region. Control of malaria encounters great challenge because of drug resistance of parasite and mosquito vector. In the beginning of 1990S, WHO/TDR suggested a new malaria control strategy by transforming the malaria-transmitting mosquito into a harmless one. In order to reach this goal, the primary basic research work is to find, cloning and study the function of the genes that support or impact the development of malaria parasites in the mosquito vector.Local melanization and encapsulation of the parasite is the most important defense response of insects, which often decide the outcome of infection. Pro-phenoloxidase (PPO) is the essential element of this defense response called PPO cascade reaction. Recognition and binding proteins, PPO and serine proteinase consist of the PPO system. Mechanical injury, chemical factor and the components of the cell wall of pathogens can activate the PPO cascade. Firstly, the PPO-activating enzyme (PPAE ) is activated by pathogen and PPAE catalyzes the transformation of PPO into the active phenoloxidase(PO) by limiting proteolysis after a small peptide was lost from PPO. PO catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine and the oxidation of dihydroxyphenylalanine and dopamine to their respective quinones, mediators of protein cross-linking, and precursors of the melanin polymer which is a central component of the melanotic capsule.Nine kinds of PPO genes have been cloned from Anopheles gambiae, 1 from Anopheles stephensi and 1 from Anopheles culicifacies to date.Anopheles dirus is the main vector of Plasmodium falciparum and plasmodium vivax in the area of Southeast Asia and the southern mountain area of China, especially in Hainan, Yunnan and Guangxi provinces. It can be infected by a kind of rodent malaria, Plasmodium yoelii, in the laboratory. However, most of oocysts were killed by melanization and encapsulation instead of maturation. So, Anopheles dirus-Plasmodium yoelii is a suitable model for study of defense mechanism of mosquito. In order to study the molecularmechanism of the defense reaction of mosquito systematically, especially the role of PPO cascade in the melanization and encapsulation of the malaria parasite, basic research work of the following aspects have been made: 1. Construction of the cDNA library from adults of Anopheles dirus. 2. Cloning of ribosomal protein S7 gene and PPOs genes of Anopheles dirus. 3. Effect of blood feeding and infection of malaria parasite on the expression of PPOs. The main experimental contents and results are as follows:1. We have got the high-quality total RNA from adult of Anopheles dirus by some methods such as cutting the head of mosquito, using more Tripure reagent and RNA precipitation buffer. On this basis, a cDNA library from adult of Anopheles dirus has been constructed. The liter of the primary library was 2.1 X 106pfu/ml and 98% of the phages were recombinants. The liter of the amplified library reaches 1X 1010- 1X 10npfu/ml. The average length of Ihe insert was above 1kb.2. According lo the conservative amino acid sequence blocks of ribosomal prolein S7 genes (S7) of human, rodenl and several insects, a degenerated primer pair was designed. Partial cDNA sequence of S7 of Anopheles dirus, designated as AdS7, was cloned. A complete cDNA sequence of AdS7 was isolated through screening the cDNA library. AdS7 has the highest sequence similarity with the S7 of Anopheles gambiae(AgS7). AdS7 is 94% identical in DNA sequence and 96% in amino acid sequence to AgS7. AdS7 was a stable internal control gene for its expression was not affected by normal blood or infective blood feeding in Anopheles dirus.3. Three cDNA sequences for Anopheles dirus PPO, designated as AdPPOl, AdPPO2 and AdPPOS, were cloned through RT-PCR using a degenerated primer pair. They are the homologues of AgPPOS, AgPPO2 and AgPPO6, respectively, according to the blast search result. A 1533bp cDNA of AdPPOl wi...