Using T7 CDNC Phage Library Screening Gene Of Interaction Protein Between Dengue Type 2 Virus And Aedes Albopictus

Objectives:1. mRNA of Ae. albopictus was first isolated from Ae. albopictus, and was then used tosynthesize double strain cDNA by the reverse transcription. Then construct T7 phage displaycDNA library from Aedes albopictus,and identify the quality of the phage display cDNAlibrary.2. Reproducing the type 2 dengue virus by intermediate vector C6 / 36 cells and purifyingdengue type 2 virus. Measure the titer of the purification of dengue type 2 virus, and theinfection of the purified dengue type 2 virus.3. Using the T7 phage display cDNA library from Aedes albopictus and the the purified denguetype 2 virus, by the menthe of the protein molecular hybridization, screen the interactingproteins between dengue viruses and Aedes albopictus in a simple. Then using the BLAST toanalyze them.Methods:1. Construction of T7 phage display cDNA library from Aedes albopictus: mRNA of Ae.albopictus was first isolated from Ae. albopictus, and was then used to synthesize double straincDNA by the reverse transcription. The double strain cDNA was attached with EcoRⅠand HindⅢadhering-ends by being ligated with the directional EcoRⅠand HindⅢlinkers. The doublestrain cDNA fragments which were longer than 300 bp in length were fractionated by the MiniColumn, and these fractionated fragments were then ligated into the T7 Select 10-3b vector withEcoRⅠand HindⅢadhering-ends. After have being packaged in vitro, the recombinant T7Select 10-3b was transformed into BLT 5 403 for constructing T7 phage display cDNA library.2. Purify Dengue Type 2 Virus: Reproducing the dengue virus type 2 by intermediate vector C6/ 36 cells, then through analyzing of the situation of the C6 / 36 cell which were infected andreplicated by dengue type 2, determine the highest titer viruses, optimal proper time period ofharvest the dengue 2 type virus, and then do a lot of multiplication of viruses by infection theC6/36 cell. After getting a large number of dengue type 2 virus liquid, freezing and thawing theliquid three times for released the viruses ,concentrated the proliferation of dengue virus frominfected C6 / 36 cells by adding PEG-8 000 in, then suspend the viruses with TNE. With 30%sucrose cushion, ultracentrifuging the suspension, the precipitation is the purification of viruses. 3. Screening the Interactional Protein between Aedes albopictus and Dengue Type 2 Virus fromthe T7 phage cDNA Library of Aedes albopictus: Using T7 phage display, and the purification ofdengue type 2 as a target molecule, the phage of Aedes albopictus cDNA library was screenedseveral rounds of biological screening, the result of the screened DNA clone sequence wasanalyzed and homology searching.Result：1. T7 phage display cDNA library from Aedes albopictus got the basic requirements of cDNAlibrary from recombination rate, the titer of library and uniformity of library. According to thetitle testing results, the titer of present library and its amplified library was 1.7×107pfu/mL and2.5×10¹³pfu/ mL respectively. PCR identification on fifty randomly selection clones was alsofinished, the PCR results showed that there was ninety-five percent of these clones wererecombinant and ninety percent such contaminant clones contained cDNA fragments that waslonger than 300 bp in length. T7 phage display cDNA library from Aedes albopictus could beenough for utilizing for identification of the gene that functions in interaction between Ae.albopictus and mosquito-born virus.2. The C6/36 cells were shrinkage, deformation after the viruses infected 5 days and then theemergence of multicellular forms, 7-8 days the emergence of a large number of cell disruption.When the cell occurs emergence of fusion, stored the cells at - 20℃. The titer of the purificationof dengue type 2 virus is TCID50 = 10-3.875. The SDS-PAGE analysis of the purification ofdengue type 2, a resistance of dengue virus band was seen obviously. Therefore, thepurification of dengue type 2 virus is contagious, which is enough for the experiment bellowing.And we also found an easy and convenient menthe of purifying of dengue type 2 virus.3.Using purified dengue type 2 virus to screen T7 phage display cDNA library from Aedesalbopictus. After identify, two positive clones were obtained, searching the sequencing results inthe NCBI EST database for homology and alignment （ BLAST ）, there is a section of Aedesaegypti salivary gland genes, some of Aedes aegypti degradation of mRNA protein gene, thesequence homology were high. Aedes aegypti and Aedes albopictus homology is high, the resultcan be identified as the dengue type 2 virus and Aedes albopictus interacting protein whichmay be located in salivary gland of Aedes albopictus, also found Aedes aegypti degradation ofmRNA protein gene which may be the dengue type 2 virus and the mosquitoes combination ofcertain relation exists. And Aedes aegypti and Aedes albopictus are homologous. So the resultcan identify interacting proteins between dengue virus and Aedes albopictus located in the salivary gland of Aedes albopictus, which is the same as the most researchers said, and the resultfound by the way of molecular biology, which is a very powerful way now. At the same time, wefound a result is same as Aedes aegypti degradation of mRNA protein gene. The result says theinteracting proteins between dengue virus and Aedes albopictus may have some relationship withthem, which also can found the evidence before.Conclusion:1. One T7 phage display cDNA library from the Ae. albopictus was constructed successfully,which satisfied the molecule library primary requirement and could be utilized for identificationof the gene that functions in interaction between Ae. albopictus and mosquito-born virus. Andthe library also can use for the further research of the mechanism of the introduction betweendengue virus and Ae. Albopictus, and it was also the basic of the research of mosquito-born virus,providing the enough material. It does a research of the spread of mosquito-born virus fromopinion of molecular biology.2. This menthe for the titers is proper for the requirements of screening the cDNA culture , thetiter purified dengue type 2 virus is proper for the requirements; the through SDS-PAGE analysisthe purification of dengue type 2 virus, these can be seen the objective band obviously,therefore, the purification of dengue type 2 virus is contagious. And the way of purifying denguetype 2 virus is effective.3．The T7 phage display system is the fast and effective means of screening of interactingproteins in a simple, the result of the interaction of the protein from screening can be use for thefurther study in the mechanism of Aedes albopictus infected with dengue 2 virus ,which is alsoprovided important basis for the mechanisms of susceptibility of the vector mosquitoes to denguevirus , understand the characteristics of the dengue fever epidemic and developing significantlyof the method of controlling arthropod-borne virus. Many papers proved that there werereceptors of dengue vires existed in the Aedes albopictus, they may located in the salivary glands,midgut, ovary and also other places. Due to the time limited, we just screening a few times, andonly screening the Aedes aegypti salivary gland genes, some of Aedes aegypti degradation ofmRNA protein gene, which will be a new way of finding the receptors of dengue virus.