The Complete Sequence Cloning And The Fragments Prokaryotic Expression,Protein Purification And Antibody Preparation Of DPV UL19 Gene

This thesis about duck plague virus CHv strain UL19 gene (GenBank Accession No. EU195092) focuses researches on the bioinformatics analysis, the complete sequence cloning and the fragments prokaryotic expression, protein purification and antibody preparation. The main experimental results are as follows:1. The bioinformatics analysis of DPV UL19 geneThe complete sequence of DPV UL19 gene is 4143 bp and encoding 1380 amino acids, whose theoretical molecular weight is 153.674 kDa. Protein similarity comparison indicates that the encoded protein is highly conservative. So we consider the DPV UL19 gene encoding the main capsid protein of this virus. Further analysis shows that the polypeptide contains no signal peptide and is not secreted proteins. This protein contains almost no transmembrane region and is preliminarily considered to be the outside membrane protein. The hydrophobic areas are widely dispersed and scattered. This polypeptide is mainly located in the nucleus, cytoplasm and endoplasmic reticulum where there is higher content. We speculate it may have something to do with capsid assembling process that the capsid is assembled within the host cell nucleus and then transferred to the cytoplasm for further processing. According to the prediction of tertiary structure, a high reliability model was found to correspond with a segment (aa 486-1050) in this peptide.2. The complete sequence cloning of DPV UL19 geneSince the complete sequence of DPV UL19 gene is a little longer, the undesirable nonspecific banding is easy to appear in PCR amplification. Therefore, it is quite hard to choose the PCR amplification condition. We send the relatively pure PCR amplification products to sequencing company which inserts this gene into the vector pUC118-2 and sequences the inserted gene. Then we construct the recombinant expression plasmid pGEX-4T-1/UL19 and use IPTG inducible expression. After transformation of this recombinant plasmid into different host bacteria and a variety of conditions, ultimately we do not get the recombinant protein which is correspond with theoretical value by washing inclusions and column purification. So we make a conclusion that this gene is difficult to be expressed in prokaryotic expression system. The possible reason is that the sequence is so long that it cannot correctly folded into the spatial structure of protein in the process of inducible expression, which lead to the interrupted expression.3. The fragments prokaryotic expression, protein purification and antibody preparation of DPV UL19 geneAccording to the antigen epitope analysis, the sequences located in middle part of this gene are more likely to have B cell antigen epitope. So we divide the middle part into three fragments and begin to T clone and subclone respectively. Then the recombinant plasmids are transformed in different host bacteria for inducible expression and three types of corresponding expression products are mainly in the form of inclusion bodies. After using a variety of purifying methods, finally we can get the more pure proteins with gel extraction and washing inclusions. The purified proteins are used as antigens for immunity to rabbits. Thus we can get the polyclonal antibodies and detect the corresponding titer by agar gel diffusion test. The titer of three prepared polyclonal antibodies are 1:8,1:16 and 1:16 respectively.