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 หัวเรื่อง:ไม่มีชื่อไทย (ชื่ออังกฤษ : Cloning and Expression of HA Gene from a Highly Pathogenic Avian Influenza Isolate (H5N1) in Thailand) ผู้เขียน: ดร.เกรียงไกร วิฑูรย์เสถียร, ผู้ช่วยศาสตราจารย์ , ดร.ธีระ รักความสุข, รองศาสตราจารย์ , ดร.ทวีศักดิ์ ส่งเสริม, ศาสตราจารย์ , ดร.ธีระพล ศิรินฤมิตร, รองศาสตราจารย์ สื่อสิ่งพิมพ์:pdf AbstractThe avian influenza A virus hemagglutinin (HA) protein is encoded by viral gene segment IV and mediates early steps of the viral replication cycle, receptor binding and membrane fusion. This research was aimed to clone and express HA gene of avian influenza virus (AIV) in insect cell cultures using a baculovirus expression vector system. The viral RNA was extracted from the allantoic fluid of 9 to 11 days old chicken embryonic eggs. The extracted viral RNA was used as template for HA gene synthesis using RT-PCR ~ 1,700 long. The amplicons were cloned into plasmid pFastBac HT and were transformed into E. coli strain DH10 Bac to produce the recombinant H5 baculovirus bacmids. The recombinant baculovirus DNA was further used to transfect and express in insect cell cultures. By SDSPAGE, the recombinant H5 protein was found to be 65 kDa in size. This protein was analyzed by dot blot and Western blotting using goat anti-H5 AIV polyclonal antibody and mouse anti-histidine monoclonal antibody. The results indicated that the HA gene was successfully cloned and the H5 protein could be expressed in insect cell cultures using a baculovirus expression vector system. Therefore, H5 protein could be further developed and applied as a candidate H5 AIV subunit vaccine. |
หัวเรื่อง:ไม่มีชื่อไทย (ชื่ออังกฤษ : Cloning and in vitro Expression of N1 Neuraminidase Gene of Avian Influenza Virus A/Duck/Thailand/KU-KPS/2004(H5N1)) ผู้เขียน:นายอลงกต บุญสูงเนิน, ผู้ช่วยศาสตราจารย์, ดร.ทวีศักดิ์ ส่งเสริม, ศาสตราจารย์, ดร.ปริวรรต พูลเพิ่ม, ผู้ช่วยศาสตราจารย์ , นางอมรา ทองปาน, รองศาสตราจารย์, ดร.ธานีรัตน์ สานติวัตร, รองศาสตราจารย์, ดร.ธีระพล ศิรินฤมิตร, รองศาสตราจารย์ สื่อสิ่งพิมพ์:pdf AbstractAvian influenza virus (AIV) can spread rapidly and causes serious disease to animals and humans so it is crucial to have effective diagnostic tools for determining its existence. The purposes of this study were first, to clone N1 neuraminidase (NA) gene of AIV A/Duck/Thailand/KU-KPS/2004 (H5N1), and second, to express recombinant N1 using baculovirus expression system. Viral RNA extracted from positive AIV allantoic fluid was reverse-transcribed to cDNA and then was amplified by PCR using N1 specific primers. The PCR products approximately 1,300 bp that encode the N1 was introduced into baculovirus vector in order to allow N1 expression in insect cell lines. Immuno-dot blot analysis of crude extract of baculovirus infected insect cells using goat-anti H5N1 AIV hyperimmune serum gave a clear immunoreactive spot. SDS-PAGE analysis showed banding patterns from the crude extract contained major protein of 54 kDa. Western blot analysis using goat-anti H5N1 hyperimmune serum and mouseantihistidine monoclonal antibody also gave a specific band approximately the same size as that of SDSPAGE. Expression analysis of this gene indicated that recombinant neuraminidase may have correct post-translation modification and folding. The recombinant neuraminidase could be very useful for the development of a test kit that can differentiate infected from vaccinated animals (DIVA). |
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