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CLONING AND SEQUENCING OF HEMAGGLUTININ GENES
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CLONING AND SEQUENCING OF HEMAGGLUTININ GENES Gritsenko D., Deryabina N., Kassenova A. Institute of Plant biology and biotechnology Institute of Plant protection and Quarantine al-Farabi Kazakh National University ninaderyabina11@gmail.com Hemagglutinin (HA) is an envelope protein of influenza A virus and the basic target molecule while developing novel medicine and vaccines. There are 18 subtypes of HA, whose hosts vary from aquatic birds, domesticated and wild mammals, bats and humans. Influenza A virus has an error-prone RNA polymerase, which introduces many mutations per replication cycle. That is why influenza A virus mutates so rapidly: hemagglutinin, neuraminidase, ion channel protein, other viral proteins obtain changes in their amino acid 151 sequence. Due to constant possibility of a novel influenza pandemics occurrence, a highly effective vaccine should be proceeding through the development to reduce both financial and medical losses. The first step in the development of a novel vaccine is cloning and sequencing of hemagglutinin gene. Firstly, total RNA was isolated from aquatic birds by a standard protocol. Then RNA of HA was used to synthesize HA’s cDNA by reverse transcription method to study and analyze its nucleotide sequence of influenza A virus. In this work we have obtained cDNA from 3 different subtypes. Complete and amplified HA genes of 3 various subtypes were cloned into T- vector, which has thymine molecules on its end to facilitate consequent cloning. T-vector allows for direct selection of target products right during amplification, that is why additional investigative methods were not required. Further, we have determined nucleotide sequences of HA genes by using standard protocols of Sanger sequencing on computational sequencer. HA genes of H1A and H3A subtypes were subcloned into viral vector based on the Grapevine virus A genome to investigate expression of HA proteins in plants. Moreover, we performed phylogenetic analysis of the H1A and H3A proteins to understand their evolutionary relationship. Scientific adviser: PhD Galiakparov N., PhD Zhussupova A. жүктеу/скачать 2,79 Mb. Достарыңызбен бөлісу:
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