臺灣博碩士論文加值系統

近年生物科技的進步，利用乳酸菌基因工程生產特殊功能的外源蛋白，在學術及商業上具有重要之價值與意義。但目前商業上能提供之基因表現載體系統不多且極為昂貴，載體穩定性及適用宿主範圍也極具改進空間。本研究室由Lactobacillus sakei分離得一6212 bp之內源質體pTHU-1。本實驗將pTHU-1經完整序列分析及同源性比對，質體上含複製起始區及十個開放閱讀區。同源性比對結果顯示，質體以θ模式進行複製；開放閱讀區之ORF1轉譯複製起始蛋白A，ORF2轉譯複製起始蛋白B，ORF3轉譯修飾蛋白HsdS，ORF6轉譯tyrosine recombinase。本實驗採用Lactobacillus sakei的啟動子Pldh連接gusA報導基因重組至pTHU-1中，建構一個可於Lactobacillus sakei表現外源蛋白的自體選殖載體pTHU-3，並探討此重組質體在Lactobacillus sakei中的表現能力。

The DNA recombination technology applied on the improvement of lactic acid bacteria (LAB) becomes more convenient in recent year. Development of heterogenous protein expression vectors for LAB has been considered as an interesting topic of academic research and with commercial potential, since there is limit available expression systems for LAB on the shelf at present.
In our laboratory, an endogenous plasmid of Lactobacillus sakei was found and named as pTHU-1. The nucleotide sequence of this plasmid has been identified, and the data have been registered in the NCBI database (No.bankkit796817). In this thesis, the nucleotide sequence has been analyzed, and the element of replication origin and ten ORFs were identified. The sequence of replication origin revealed that pTHU-1 replicates by theta mode. Among the ORFs ORF1, ORF2, ORF3, and ORF6 were recognized encoding a Rep A protein, a Rep B protein, a type I restriction-modification system specificity subunit (HsdS), and a tyrosine recombinase respectively. On the other hand, making use of pTHU-1 to develop a self-cloning vector was undertaken. The gusA gene served as a selection marker with the promoter Pldh of ldh (lactate dehydrogenase) from Lactobacillus sakei was recombined with pTHU-1 to form a expressed plasmid, pTHU-3. The recombinant plasmid was introduced into Lactobacillus sakei and some other LABs, but the expression was not significant yet.

中文摘要 I
英文摘要 II
壹、前言 1
貳、文獻回顧 2
一、 乳酸菌表現載體之開發 2
(一) 乳酸菌之內源質體 2
(二) 質體複製模式 3
(三) 控制表現系統 14
(四) 乳酸菌常用之選殖標記系統 18
(五) 乳酸菌建構載體之轉形作用及宿主選擇 20
(六) 乳酸菌之表現系統及應用 22
二、 利用Lactobacillus sakei內源質體開發表現載體 26
(一) Lactobacillus sakei基本性質 26
(二) Lactobacillus sakei於基因工程上的發展及應用 27
參、材料與方法 32
一、 試驗材料 32
(一) 菌種及質體 32
(二) 培養基 36
(三) 儀器設備 37
二、 實驗方法 37
(一) 菌種活化及保存 37
(二) 質體DNA快速抽取法 38
(三) 乳酸菌染色體快速抽取法 39
(四) 構築表現載體 40

肆、結果與討論 46
一、 質體DNA之篩選及限制酶截切作用 46
(一) pEZZ18 46
(二) pGPTV-KAN 46
(三) pTHU-1 47
二、 乳酸菌質體序列分析 51
(一) pTHU-1之限制酶圖譜分析 51
(二) pTHU-1之開放閱讀區核酸序列分析 55
(三) pTHU-1之複製起始點核酸序列分析 58
三、 利用pTHU-1建構乳酸桿菌表現載體 67
(一) 報導基因gusA及其啟動子Pldh之製備 67
(二) Pldh::gusA選殖至大腸桿菌系統 69
四、 pTHU-3建構與自體轉殖 85
伍、結論 91
陸、參考文獻 92
附錄一、pTHU-2建構流程圖 99
附錄二、pEZZ18-LDH以M13 forward定序圖譜 100
附錄三、pEZZ18-LDH以M13 reverse定序圖譜 101
附錄四、pEZZ18-GUS以M13 forward定序圖譜 102
附錄五、pEZZ18-GUS以M13 reverse定序圖譜 103
附錄六、pEZZ18-LDHGUS以M13 forward定序圖譜 104
附錄七、pEZZ18-LDHGUS以M13 reverse定序圖譜 105


表次
表一、乳酸菌之滾動環狀型複製質體 5
表二、乳酸菌之θ型複製質體 10
表三、θ型複製之分類 13
表四、乳酸菌之誘導表現系統 15
表五、乳酸菌之醣類誘導表現系統 16
表六、乳酸菌之質體大小及其分佈情形 21
表七、Lactococcus lactis轉錄起始區序列 23
表八、乳酸球菌轉譯起始區之核醣體結合位 24
表九、乳酸菌中異源基因表現實例 25
表十、L. sakei細菌素之生化特性及抑制菌屬 29
表十一、pTHU-1單一限制酶截切點 52
表十二、pTHU-1開放閱讀區之基本特色及產物 59


圖次
圖一、RCR質體之複製模式 7
圖二、θ型複製質體之複製模式 12
圖三、乳酸菌之誘導基因表現系統 17
圖四、基質X-Gluc經β-glucuronidase分解產生glucuronic acid及5-bromo-4-chloro -3-indolyl 19
圖五、Lactobacillus sakei之細菌素基因 30
圖六、剔除Lactobacillus sakei 23K轉譯β-galactosidase的lacLM 操縱元 31
圖七、質體pGPTV-KAN之限制酶圖譜 34
圖八、質體pEZZ18之限制酶圖譜 35
圖九、不同限制酶截切pEZZ18之洋菜膠電泳分析圖 48
圖十、不同限制酶截切pGPTV-KAN之洋菜膠電泳分析圖 49
圖十一、不同限制酶截切pTHU-1之洋菜膠電泳分析圖 50
圖十二、pTHU-1以單一限制酶截切之洋菜膠電泳分析圖 53
圖十三、質體pTHU-1開放閱讀區及限制酶圖譜 54
圖十四、pTHU-1之ORF1與其他乳酸菌相似胺基酸序列比對 61
圖十五、pTHU-1之ORF2與其他乳酸菌相似胺基酸序列比對 62
圖十六、pTHU-1之ORF3與其他乳酸菌相似胺基酸序列比對 63
圖十七、pTHU-1之ORF6與其他乳酸菌相似胺基酸序列比對 64
圖十八、pTHU-1之複製起始區核酸序列分析 65
圖十九、(A) pRV500與 (B) pTHU-1的複製起始區結構圖 66
圖二十、Lactobacillus sakei聚合酶連鎖反應之Pldh基因產物分析電泳圖 68
圖二十一、預期成功轉型菌株之質體 (A) pEZZ18-LDH (B) pEZZ18-GUS (C) pEZZ18-LDHGUS基因圖譜 71
圖二十二、轉形株之藍白篩選 72

圖二十三、以不同限制酶截切pEZZ18-LDH、pEZZ18-GUS及pEZZ18-LDHGUS之電泳圖 73
圖二十四、重組質體Pldh序列之聚合酶連鎖反應之產物電泳圖 74
圖二十五、pEZZ18-LDH定序結果 77
圖二十六、pEZZ18-LDH之Pldh序列與L. sakei 23K之Pldh基因序列比對結果 78
圖二十七、pEZZ18-GUS定序結果 (A) 以引子M13 forward定序 (B) 以引子M13 reverse定序 79
圖二十八、pEZZ18-GUS以 (A) 引子M13 forward (B) 引子M13 reverse定序之gusA序列與E. coli之uidA序列比對結果 81
圖二十九、pEZZ18-LDHGUS定序結果 82
圖三十、pEZZ18-LDHGUS之 (A) Pldh 和 (B) gusA基因序列比對結果 83
圖三十一、轉殖pEZZ18-LDHGUS之大腸桿菌株於LB/Amp/X-Gluc培養基生長情形 84
圖三十二、pTHU-3建構流程圖 86
圖三十三、pTHU-3限制酶圖譜 87
圖三十四、乳酸菌於位相差顯微鏡下的型態 89
圖三十五、抽取不同乳酸菌之內源質體之洋菜膠電泳分析圖 90

Alegre, M.T., Rodriguez, M.C., and Mesas, J.M. (2004) Transformation of Lactobacillus plantarum by electroporation with in vitro modified plasmid DNA. FEMS Microbiol Lett 241: 73-77.

Alpert, C.A., Crutz-Le Coq, A.M., Malleret, C., and Zagorec, M. (2003) Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli. Appl Environ Microbiol 69: 5574-5584.

Benachour, A., Frere, J., Flahaut, S., Novel, G., and Auffray, Y. (1997) Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315. Mol Gen Genet 255: 504-513.

Bermudez-Humaran, L.G., Langella, P., Miyoshi, A., Gruss, A., Guerra, R.T., Montes de Oca-Luna, R., and Le Loir, Y. (2002) Production of human papillomavirus type 16 E7 protein in Lactococcus lactis. Appl Environ Microbiol 68: 917-922.

Bermudez-Humaran, L.G., Langella, P., Cortes-Perez, N.G., Gruss, A., Tamez-Guerra, R.S., Oliveira, S.C., Saucedo-Cardenas, O., Montes de Oca-Luna, R., and Le Loir, Y. (2003) Intranasal immunization with recombinant Lactococcus lactis secreting murine interleukin-12 enhances antigen-specific Th1 cytokine production. Infect Immun 71: 1887-1896.

Biet, F., Cenatiempo, Y., and Fremaux, C. (2002) Identification of a replicon from pTXL1, a small cryptic plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and development of a food-grade vector. Appl Environ Microbiol 68: 6451-6456.

Bruand, C., Le Chatelier, E., Ehrlich, S.D., and Janniere, L. (1993) A fourth class of theta-replicating plasmids: the pAMβ1 family from gram-positive bacteria. Proc Natl Acad Sci U S A 90: 11668-11672.

Bruno-Barcena, J.M., Azcarate-Peril, M.A., Klaenhammer, T.R., and Hassan, H.M. (2005) Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri. FEMS Microbiol Lett 246: 91-101.

Champomier-Verges, M.C., Chaillou, S., Cornet, M., and Zagorec, M. (2002) Erratum to "Lactobacillus sakei: recent developments and future prospects" [Research in Microbiology 152 (2001) 839]. Res Microbiol 153: 115-123.

Chang, S.M., and Yan, T.R. (2007) DNA sequence analysis of a cryptic plasmid pL2 from Lactococcus lactis subsp. lactis. Biotechnol Lett 29: 1519-1527.

Chin, S.C., Abdullah, N., Siang, T.W., and Wan, H.Y. (2005) Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken. J Microbiol 43: 251-256.

Danielsen, M. (2002) Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure. Plasmid 48: 98-103.

de Vos, W.M. (1999) Gene expression systems for lactic acid bacteria. Curr Opin Microbiol 2: 289-295.

Diep, D.B., Axelsson, L., Grefsli, C., and Nes, I.F. (2000) The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system. Microbiology 146 ( Pt 9): 2155-2160.

Enouf, V., Langella, P., Commissaire, J., Cohen, J., and Corthier, G. (2001) Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic. Appl Environ Microbiol 67: 1423-1428.

Fitzgerald, G.F., and Gasson, M.J. (1988) In vivo gene transfer systems and transposons. Biochimie 70: 489-502.
Gasson, M.J. (1990) In vivo genetic systems in lactic acid bacteria. FEMS Microbiol Rev 7: 43-60.

Gory, L., Montel, M.C., and Zagorec, M. (2001) Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products. FEMS Microbiol Lett 194: 127-133.

Gosalbes, M.J., Esteban, C.D., Galan, J.L., and Perez-Martinez, G. (2000) Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei. Appl Environ Microbiol 66: 4822-4828.

Gravesen, A., Josephsen, J., von Wright, A., and Vogensen, F.K. (1995) Characterization of the replicon from the lactococcal theta-replicating plasmid pJW563. Plasmid 34: 105-118.

Hazebrouck, S., Pothelune, L., Azevedo, V., Corthier, G., Wal, J.M., and Langella, P. (2007) Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei. Microb Cell Fact 6: 12.

Hertel, C., Schmidt, G., Fischer, M., Oellers, K., and Hammes, W.P. (1998) Oxygen-dependent regulation of the expression of the catalase gene katA of Lactobacillus sakei LTH677. Appl Environ Microbiol 64: 1359-1365.

Horn, N., Wegmann, U., Narbad, A., and Gasson, M.J. (2005) Characterisation of a novel plasmid p9785S from Lactobacillus johnsonii FI9785. Plasmid 54: 176-183.

Iftode, C., Daniely, Y., and Borowiec, J.A. (1999) Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol 34: 141-180.

Khan, S.A. (1997) Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev 61: 442-455.

Khan, S.A. (2005) Plasmid rolling-circle replication: highlights of two decades of research. Plasmid 53: 126-136.

Kiatpapan, P., Yamashita, M., Kawaraichi, N., Yasuda, T., and Murooka, Y. (2001) Heterologous expression of a gene encoding cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters. J Biosci Bioeng 92: 459-465.

Kiewiet, R., Bron, S., de Jonge, K., Venema, G., and Seegers, J.F. (1993) Theta replication of the lactococcal plasmid pWVO2. Mol Microbiol 10: 319-327.

Kok, J., van der Vossen, J.M., and Venema, G. (1984) Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol 48: 726-731.

Lauret, R., Morel-Deville, F., Berthier, F., Champomier-Verges, M., Postma, P., Ehrlich, S.D., and Zagorec, M. (1996) Carbohydrate Utilization in Lactobacillus sake. Appl Environ Microbiol 62: 1922-1927.

Lee, J.H., and O'Sullivan, D.J. (2006) Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector. Appl Environ Microbiol 72: 527-535.

Malleret, C., Lauret, R., Ehrlich, S.D., Morel-Deville, F., and Zagorec, M. (1998) Disruption of the sole ldhL gene in Lactobacillus sakei prevents the production of both L- and D-lactate. Microbiology 144 ( Pt 12): 3327-3333.

Martin, M.C., Alonso, J.C., Suarez, J.E., and Alvarez, M.A. (2000) Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination. Appl Environ Microbiol 66: 2599-2604.

Mathiesen, G., Huehne, K., Kroeckel, L., Axelsson, L., and Eijsink, V.G. (2005) Characterization of a new bacteriocin operon in sakacin P-producing Lactobacillus sakei, showing strong translational coupling between the bacteriocin and immunity genes. Appl Environ Microbiol 71: 3565-3574.

Meijer, W.J., Venema, G., and Bron, S. (1995) Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis. Nucleic Acids Res 23: 612-619.

Moscoso, M., del Solar, G., and Espinosa, M. (1995) In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein. J Bacteriol 177: 7041-7049.

Nes, I.F., Diep, D.B., Havarstein, L.S., Brurberg, M.B., Eijsink, V., and Holo, H. (1996) Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuwenhoek 70: 113-128.

Perez-Arellano, I., Zuniga, M., and Perez-Martinez, G. (2001) Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli. Plasmid 46: 106-116.

Pillidge, C.J., Cambourn, W.M., and Pearce, L.E. (1996) Nucleotide sequence and analysis of pWC1, a pC194-type rolling circle replicon in Lactococcus lactis. Plasmid 35: 131-140.

Platteeuw, C., van Alen-Boerrigter, I., van Schalkwijk, S., and de Vos, W.M. (1996) Food-grade cloning and expression system for Lactococcus lactis. Appl Environ Microbiol 62: 1008-1013.

Raha, A.R., Hooi, W.Y., Mariana, N.S., Radu, S., Varma, N.R., and Yusoff, K. (2006) DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14. Plasmid 56: 53-61.

Ribeiro, L.A., Azevedo, V., Le Loir, Y., Oliveira, S.C., Dieye, Y., Piard, J.C., Gruss, A., and Langella, P. (2002) Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis. Appl Environ Microbiol 68: 910-916.

Rochat, T., Gratadoux, J.J., Gruss, A., Corthier, G., Maguin, E., Langella, P., and van de Guchte, M. (2006) Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk. Appl Environ Microbiol 72: 5143-5149.

Seegers, J.F., van Sinderen, D., and Fitzgerald, G.F. (2000) Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain. Microbiology 146 ( Pt 2): 435-443.

Serror, P., Sasaki, T., Ehrlich, S.D., and Maguin, E. (2002) Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids. Appl Environ Microbiol 68: 46-52.

Shareck, J., Choi, Y., Lee, B., and Miguez, C.B. (2004) Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology. Crit Rev Biotechnol 24: 155-208.

Skaugen, M., and Nes, I.F. (2000) Transposition in Lactobacillus sakei: inactivation of a second lactocin S operon by the insertion of IS1520, a new member of the IS3 family of insertion sequences. Microbiology 146 ( Pt 5): 1163-1169.

Sorensen, K.I., Larsen, R., Kibenich, A., Junge, M.P., and Johansen, E. (2000) A food-grade cloning system for industrial strains of Lactococcus lactis. Appl Environ Microbiol 66: 1253-1258.

Sorvig, E., Gronqvist, S., Naterstad, K., Mathiesen, G., Eijsink, V.G., and Axelsson, L. (2003) Construction of vectors for inducible gene expression in Lactobacillus sakei and L. plantarum. FEMS Microbiol Lett 229: 119-126.

Sorvig, E., Mathiesen, G., Naterstad, K., Eijsink, V.G., and Axelsson, L. (2005) High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology 151: 2439-2449.

Stentz, R., Loizel, C., Malleret, C., and Zagorec, M. (2000) Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB. Appl Environ Microbiol 66: 4272-4278.

Sudhamani, M., Ismaiel, E., Geis, A., Batish, V., and Heller, K.J. (2007) Characterisation of pSMA23, a 3.5kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus. Plasmid.

Takala, T.M., Saris, P.E., and Tynkkynen, S.S. (2003) Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG. Appl Microbiol Biotechnol 60: 564-570.

te Riele, H., Michel, B., and Ehrlich, S.D. (1986a) Are single-stranded circles intermediates in plasmid DNA replication? Embo J 5: 631-637.
te Riele, H., Michel, B., and Ehrlich, S.D. (1986b) Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc Natl Acad Sci U S A 83: 2541-2545.

Thompson, J.K., Foley, S., McConville, K.J., Nicholson, C., Collins, M.A., and Pridmore, R.D. (1999) Complete sequence of plasmid pLH1 from lactobacillus helveticus ATCC15009: analysis reveals the presence of regions homologous to other native plasmids from the host strain. Plasmid 42: 221-235.

Viegas, S.C., Fernandez De Palencia, P., Amblar, M., Arraiano, C.M., and Lopez, P. (2004) Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis. Plasmid 51: 256-264.

Walker, S.A., and Klaenhammer, T.R. (1998) Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage phi31. J Bacteriol 180: 921-931.

Wang, T.T., and Lee, B.H. (1997) Plasmids in Lactobacillus. Crit Rev Biotechnol 17: 227-272.

Wu, C.M., Lin, C.F., Chang, Y.C., and Chung, T.C. (2006) Construction and characterization of nisin-controlled expression vectors for use in Lactobacillus reuteri. Biosci Biotechnol Biochem 70: 757-767.

Zhou, X.X., Li, W.F., Ma, G.X., and Pan, Y.J. (2006) The nisin-controlled gene expression system: construction, application and improvements. Biotechnol Adv 24: 285-295.

Zuniga, M., Pardo, I., and Ferrer, S. (1996) Nucleotide sequence of plasmid p4028, a cryptic plasmid from Leuconostoc oenos. Plasmid 36: 67-74.