DEVELOPMENT OF BICISTRONIC BACULOVIRUS EXPRESSION SYSTEM VECTOR USING BAC TO BAC SYSTEM (tt)

摘要 桿狀病毒的研究著重在昆蟲病毒的基本病毒學與生物技術之應用。桿 狀病毒表達載體系統（BEVS）是以重組的昆蟲桿狀病毒感染昆蟲細胞來 表現蛋白質，是分子生物學的一種重要工具。穿梭桿狀病毒表達載體可以 發揮哺乳動物細胞與昆蟲細胞的功能, 讓他們的發展會得到一個重要的優 勢。我們把內部核糖體進入位點（IRES）跟桿狀病毒基因組連結在一起， 然後生成可同時表達兩種基因之雙順反子桿狀病毒表達載體。在這個報告 中，我們試著建造一個桿狀病毒載體擁有使用 Bac-to-Bac 系統的依雙順反 子表達載體之 IRES。傳統方法使用在昆蟲細胞的同源重組, 不過 Bac-toBac 系統帶來比傳統方法更多的好處。Bac-to-Bac 質粒: pFastBac1-PolhMCS-Lir-DsRed2 已成功生成。這種 IRES- 基於 Bac-to-Bac BEVS 將促進 快速和有效地產生重組桿狀病毒：節省時間表達桿狀病毒質粒，一步純化 和擴增，多重組病毒的快與和同時隔離以及滴度測定。 關鍵詞：桿狀病毒、桿狀病毒表達系統、Bac-to-Bac 系統、重組蛋白 i Abstract Baculoviruses are one of the most studies insect viruses both in basic virology research and in biotechnology applications This insect viruses based baculovirus expression vector system (BEVS) is an important tool for molecular biology and widely used in insect cells Development of a shuttle baculovirus expression vector which can function in both the mammalian cell as well as in insect cell would have a significant advantages We had cooperated an internal ribosome entry site (IRES) in to the baculovirus genomes and generated a bicistronic baculoviruses expression vector that can express two genes simultaneously In this project, we attempt to construct a baculovirus vector with an IRES based bicistronic expression vector which using Bac-to-Bac system The Bac-toBac system bring more benefits than the traditional methods using homologous recombination in insect cells Bac-to-Bac plasmids: pFastBac1-Polh-MCS-Lir-DsRed2 had been successfully generated Such IRES-based Bac-to-Bac BEVS will facilitate rapid and efficient generation of recombinant baculoviruses: time saving expression bacmid, one-step purification and amplification, rapid and simultaneous isolation of multiple recombinant viruses as well as titer determination Key words: Baculovirus, Baculovirus expression system, Bac-to-Bac system, recombinant protein ii Acknowledgements There are many people I would like to acknowledge whose support has made this dissertation possible First of all, I would to thank my advisor, Prof Wu Tzong-Yuan, who has guided and supported me I am very grateful for the opportunity he gave me to develop research and intellectual skills in his lab and to share in his wealth of experience and knowledge Besides my advisor, the good advice, assistances in the research, support, and friendship from Cheng Ying-Yu (Assam), Joyce Teng, Y-Ting Lin have been invaluable on both an academic and a personal level, for which I am extremely grateful In addition, I thank my fellow lab mates: Mato, Liu Ming-Kun, Johny at Genetics Engineering Lab in Bioscience Department, CYCU My appreciation also goes to all of the Bioscience Technology faculty and staff I have enjoyed my coursework which would not be possible without all the work you I would also like to express my thanks for the ceaseless support, guidance, and friendships of Prof Chan, Dr Chen, Dr Chin, Dr Hsu, Dr Chiao You all have made this experience enjoyable for me Living in Taiwan gave me many wonderful experiences not only in my research but also in my life, so I would like to acknowledge the financial, academic and technical support of CYCU, and its staff, particularly in the award of a Distinguished International Graduate Students that provided the necessary financial support for this master degree in CYCU and living cost in Taiwan for two years iii Finally, I would like to thank my parents, my older sister, my friend and fellowship for their prayers and encouragement me both in intellectual and materials I could not have gone this far without their love and support iv Contents 摘要 i Abstract .ii Acknowledgements iii Contents .v Table List .viii Figure List .ix The aim of study………………………………………………… …………………… x Chapter Introduction and Literature Review 1.1 Introduction .1 1.2 Baculovirus…………… 1.3 Baculovirus replication cycle 1.4 Baculovirus expression vector system (BEVS) … .5 1.5 Recombinant Baculovirus production 1.6 Promoters used for the expression of foreign gene .8 1.7 Internal ribosome entry site (IRES) 1.8 Bac-to-Bac expression vector system………………… …………………11 v Chapter Materials and Methods 14 2.1 Materials 14 2.1.1 Virus, cell and transfection 14 2.1.2 Chemicals and reagents 14 2.1.3Instruments………………… … 15 2.2 Methods…………… 16 2.2.1Competent cell 16 2.2.2 Transformation 17 2.2.2.1Transformation with DH5α competent cells……… … … 17 2.2.2.2 Transformation with DH10Bac competent cells… … …… …19 2.2.3 Plasmid DNA extraction .19 2.2.4 DNA electrophoresis 20 2.2.5 DNA gel extraction 21 2.2.6 Ligation 21 2.2.7 Restriction enzyme digestion………… .22 2.2.8 Vector construction 22 2.2.9 Bac-to-Bac expression system…………………………… … 23 vi 2.2.10 Transfection……………………… …………………………… ….24 2.2.11Recombinant virus production and titer determination……… … ….24 2.2.12 End-point dilution…………………………………… ………….….25 2.2.13 Insect cell lysis………………………………………….………….…26 2.2.14 Protein concentration detection…………………………… …… …26 2.2.15Western blot analysis……………….……………….… ………….…26 Chapter Results and discussions 29 A- Results……………………………………………………………………… …29 3.1 Vector construction……………………………………….………… …29 3.2 Recombinant baculovirus……………………………………….….… 29 3.3 Transfection of Sf21 insect cells with the recombinant-bacmid DNA….30 3.4 Expression of Red fluorescent protein by Recombinant baculovirus in insect cells infected by recombinant baculovirus…………… …….………………… 30 B- Discussions…………………………………………………………………….…31 Conclusions 35 References 36 vii Table List Table The composition of PBS and reagents used in Western blot……………………43 Table 2.M13/pUC Amplification primers for the analysis of recombinant bacmid DNA.45 viii Figure List Figure 1.Forms of baculovirus virion-OV (occluded virus) and BV (budded virus)…46 Figure 2.Budded virus replication cycle …………………………………………… 47 Figure 3.Diagram of Bac-to-Bac system …………………………………………… 49 Figure 4.Outline of Bac-to-Bac system proceduce……………………………………50 Figure 5.Research design………………………………………………………… .51 Figure6.Construction of pFastBac™1 vector ……………………………………… 52 Figure 7.Circular map of pFastBac1-Polh-MCS-Lir-DsRed2……………………… 53 Figure8.Analysis construction of bi-cistronic pFastBac1-Polh-MCS-Lir-DsRed2 vector by restricton enzyme ………………………………………………………………….…54 Figure 9.Construction of the recombinant vAc-MCS-Lir-DsRed2 virus…………….55 Figure 10 PCR analysis using M13 primer to confirm the size of bacmid pFastBac1-PolhMCS-Lir-Dsred2………………………………………………………………… 56 Figure 11.Generation of recombinant virus vAc-MCS-Lir-DsRed2 expressing protein in Sf21 cell lines……………………………………………………………………… 57 Figure 12.Analyzing protein expression in time course from 1-6 days after infection with different m.o.i by Western Blot using the 1st Ab anti-RFP (1:1000), 2nd Ab anti – HRP (1:5000)…………………………………………………………………………… 59 ix The aim of study Bac-to-Bac Baculovirus Expression system is a rapid and efficiency method to generate recombinant baculovirus It is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector propagated in E.coli Using sitespecific transposition to insert foreign genes into a bacmid propagated in E coli has a number of advantages over the generation of recombinant baculoviruses in insect cells by homologous recombination Recombinant virus DNA isolated from selected colonies is not mixed with parental, non-recombinant virus, eliminating the need for multiple rounds of plaque purification Expression of two genes in one cell achieved by coinfection with several recombinant viruses, each carrying a single foreign gene However, some studies have shown that infection with one baculovirus containing all heterologeous genes can results in up to 30-fold protein yields compared to multiple single-gene virus The mutated DNA construct will be transformed into DH10Bac cells for generating bacmid DNA, which will be isolated and analyzed by agarose gel electrophoresis and PCR Insect cells will be transfected with the recombinant bacmid DNA to produce mutant proteins which will further be purified and analyzed Perhaps the greatest advantage of this method is that it permits the rapid and simultaneous isolation of multiple recombinant viruses, and is particularly suited for the expression of protein variants for structure/function studies The construction of bicistronic transfer vectors for the baculovirus expression system (BEVS), which are advantageous over the existing vectors The new vectors provide a simple way to isolate recombinant viruses x This study aimed to use Bac-to-Bac expression system to expression high protein in bi-cistronic vector because the recombinant protein are usually produced by replacing the viral polyhedrin gene with a foreign encoding for the protein of interest xi Chapter Introduction and Literature Review 1.1 Introduction It is well known that baculoviruses are generally considered to be beneficial to humans (Carstens, 1980) The journey into unraveling the importance of baculoviruses to human beings began with studies that explored the wilting disease of silkworm (Benz, 1986; Basil, 2005) by Marco Vida of Cremona, an Italian bishop in the 16th century Several lines of evidence indicate that the biological importance of baculoviruses has been increasing since the discovery of its biopesticide potential in crop fields in the 1940 A unique feature of this discovery is the ability of each virus species to cause disease in only one or a small number of closely related host insect species Owing to this narrow host specificity, baculoviruses are employed as selective agricultural biological insecticides (Szewczyk et al., 2006; Inceoglu et al., 2001) Despite the increase in the biological importance of baculoviruses, there are a few shortcomings For instance, prolongation of the lavae stage as a result of the inactivation of the homone ecdysone, which is responsible for the molting of the larvae, causes the infected larvae to feed more, and subsequently leads to more crop damage before they eventually die (Szewczyk et al., 2006) In many cases, it takes 5-7 days after infection before the larvae died Thus, the use of baculovirus as a biological pesticide has been limited because of its low virulence, long killing time and its narrow host range Several measures have been taken to enhance baculovirus biopesticide performance, including introduction of insect specific hormones or toxin genes into original ... based bicistronic expression vector which using Bac- to -Bac system The Bac- toBac system bring more benefits than the traditional methods using homologous recombination in insect cells Bac- to -Bac. .. in to the baculovirus genomes and generated a bicistronic baculoviruses expression vector that can express two genes simultaneously In this project, we attempt to construct a baculovirus vector. .. construction of bicistronic transfer vectors for the baculovirus expression system (BEVS), which are advantageous over the existing vectors The new vectors provide a simple way to isolate recombinant