摘要
昆虫细胞作为重组病毒的受体能成功高效地表达外源基因，生产具有重要医用价值、具备天然活性的生物工程制品，从而使昆虫细胞培养倍受青睐。目前，可以通过转基因的方法改良现有细胞，从而获得高产昆虫杆状病毒和外源蛋白的昆虫细胞系。
本研究用磷酸钙转染法和电穿孔两种方法，分别将SV40病毒的大T抗原基因转入第80代的Tn5B1-4细胞，经G418筛选，克隆，共得到转基因细胞克隆株62株。通过细胞传代，选出生长且状况良好的克隆株共有54株。54株细胞系分别经PBS，AMD处理，病毒滴度和多角体产量比较筛选出10个细胞克隆株：Tn5B-1-1、Tn5B-1-12、Tn5B-1-15、Tn5B-1-25、Tn5B-1-26、Tn5B-1-30、Tn5B-1-33、Tn5B-2-3、Tn5B-2-9及Tn5B-2-12，这些细胞系分别接种昆虫病毒TnGV、TnNPV、MsNPV、PiNPV，其中仅Tn5B-1-12对所测的所有NPV病毒敏感，将此转基因细胞系命名为Tn5B-SV-40(简写为Tn5B-40)。
Tn5B-40细胞形态主要为纺锤形和圆形，贴壁生长，大小为18.5×42μm。增殖很快，群体倍增时间为22小时，最大生长密度可达2.2×10~6cells／ml。对AcMNPV病毒感染率达95％，多角体产量平均为115 OBs／cell，是一株高产杆状病毒的细胞系。
Tn5B-40有较强的营养抗逆性，细胞在无营养的PBS盐缓冲液中1天以后，Tn5B-40细胞有70％存活，比野生型Tn581-4细胞高出30％，第5天时，Tn581-4细胞全部死亡，Tn5B-40到第6天仍然有5％存活细胞。
用含不同浓度放线菌素D的培养基处理Tn5B-40细胞1小时，当放线菌素D浓度为0.31，0.62，1.25和2.5μg／ml时，Tn5B-40细胞的存活率分别比Tn581-4高出38％，50％，60％和35％，当浓度高达5μg／ml时，Tn581-4细胞全部死亡而Tn5B-40细胞仍有30％的生存率。说明此细胞具有抗凋亡的特性。
Tn5B-40是一株高表达重组蛋白的昆虫细胞系。用重组病毒AcMNPV-β-gal侵染细胞，Tn5B-40细胞内β-半乳糖苷酶的产量明显高于野生型细胞系，到第六天，其表达水平达到Tn581-4的2.3倍，第8天时比Tn581-4细胞高出35％，达3.4×10~4IU／ml。随着时间的延长，Tn5B-40培养基中酶量也逐渐增加，第4天就比第2天提高了5倍。第8天时Tn5B-40细胞内和培养基中的酶总表达水平达到最高5.4×10~4IU／ml。AcMNPV-SEAP感染此细胞，其细胞内的分泌型碱性磷酸酶表达水平明显低于培养基中的表达水平，最高只达1 IU／ml。Tn5B-40细胞分泌在培养基中的SEAP在细胞培养后期较高，第9天时比Tn581-4高出25％。Tn5B-40的SEAP总表达水平最高值达到5.7IU／ml。
昆虫病毒AcMNPV在Tn5B-40细胞中离体传代10次，病毒未出现继代效
应，其病毒滴度、多角体形态及产量、杀虫毒力均无明显变化。Tn5B一40细胞是
昆虫杆状病毒离体复制的优良细胞系。
To generate stable cell lines with the SV40 large T(LT) antigen, Tn5B1-4 cells was transfected with plasmids PIE-SV-LT and pAciel-Neo by using Ca3(PO4)2 and electroporation methods at passage 80. The transfected cells was firstly selected in G418-containing medium. We obtained 62 clones from the first selection after cell clone. After several passages, 55 of cell clones were examined for their resistance to nutritional stress in PBS and AMD(1μg/ml). 10 clones which appeared strong resistance to PBS or AMD and high products of OBs were obtained. Finally one of the ten clones can support the replication of three viruses: TnNPV, MsNPV and PiNPV and was named as Tn5B-SV-40 (Tn5B-40).
The morphology of Tn5B-40, grown in TNM-FH medium, generally consisted of spindle cells with presence of some round cells. The combined size of these cells was 18.5×42 μm. The Tn5B-40 cell line reached maximum density of 2.2×106cells/ml at four days and the population doubling time is 22h. Wild-type AcMNPV (MOI: 10) infected the Tn5B-40 cells with an infection of 95% of the cells and the production of occlusion bodies (OBs) by it was high, average of 115 OBs/cell.
Tn5B-40 cells showed high resistance to nutrient stress in comparison with parental cell line Tn5B1-4 when cells were exposed to PBS. Cell viability was 70% with Tn5B-40 after one day in PBS. 6 days later, 5% Tn5B-40. cells were survived but Tn5B1-4 died within five days.
When the cells were exposed to Actinomycin D concentrations of 0.31, 0.62, 1.25 and 2.5μg/ml for one hour, cell viability for Tn5B-40 was more than Tn5B1-4 cells. At a concentration of 5 μg/ml that all the Tn5Bl-4 cells were killed, but 30% of Tn5B-40 cells were still viable. It was demonstrated that Tn5B-40 cells were resistant to apoptosis induced by Actinomycin D.
Tn5B-40 cells with expressed higher level of recombinant proteins expression in comparison to its parental Tn5B1-4 cells. At six days post infection (p.i.), Tn5B-40 expressed 2.3-fold more beta-gal than Tn5B1-4 in the cells, and at eight days p.i. it produced 3.4×104IU/ml, 35% more than the parental cells. With the time of being infected by virus going, the recombinant protein in Tn5B-40 cells
medium was improved gradually and the production of β -gal at 4th days p.i. was 5-fold more than 2nd days p.i.. The total P -gal expression level in cells and medium was highest at the 8th days p.i., 5.4 X 104IU/ml. Similarly, Tn5B-40 produced significantly more secreted SEAP than Tn5Bl-4 cells. The SEAP expression level in the cells was relatively higher than in parental cells. The Tn5B-40 cells produced 25% more SEAP in medium than parental cells at the nine days p.i..At nine days p.i., the total expression level of SEAP was 5.7IU/ml.
The first serial passage of AcMNPV, which was obtained from the hemolymph of infected larvae, was established following infection of the cells at a MOI = 10. AcMNPV was replicated in Tn5B-40 cells for 10 passages continuously and there was no significant change in morphology of the occlusion bodies, OBs production and virus titer after serial passage of the virus. Bioassay was performed using OBs generated from different viral passage to examine the relationship between mortality and passage number. Under the same OBs concentration, the AcMNPV produced constant larval mortality.
引文
[1] 张浩．牛血清在细胞培养中的作用与质量要求[J]．科技情报开发与经济，2001，11(3)：46～47．
[2] 陈晓红 陈广文．我国已建立的昆虫细胞系及其生物学特性与应用[J]．昆虫知识，1996，36(4)：233-238．
[3] Kar Maramosch. Advances in cell culture[J]. 1982, 13～14.
[4] Goldshmidt R. Proc Natl Acad Sci USA[C]. 1915, 1: 220～222.
[5] Grace TDC. Establishment of four strains of cells from insect tissue grown in vitro[J]. Nature, 1962, 195: 788～789.
[6] DWIGHT E LYNN. Development and characterization of insect cell lines[J]. Cytotechnology, 1996,20: 3～11.
[7] Gharino L A,Summers MD. Nucleotide sequence and temporal expression of a baculovirus regulatory gene [J]. J. Virol, 1987, 61: 2091～2099.
[8] Jarvis D L,OkerBlom C,Summers MD. The role of glycosylation in the transport of foreign glycoproteins through the secretary pathway of Lepidopteran insect cells [J]. Cell Biochem., 1990, 42: 181～191.
[9] Hollister J R, Jarvis D I. Engineering lepidopteran insect cell for sialogly-coprotein production by genetic transfortmation with mammalian beta 1,4- galactosyltransferase and alpha 2,6-sialytransferase genes[J]. Glycobiology, 2001, 11: 1～9.
[10] Breitbach K, Jarvis D L. Improved glycosylation of foreign protein by TN-5B1-4cells engineered to express mammalian glycosyltransferases[J]. Biotechnol Bioeng, 2001, 74: 230-239.
[11] Granados R R. Status and improvements in insect cell culture for protein production[A].Prospects for the Development of Insect Factories[M]. Japan; Institute of Insect and Animal Science National Institute of Agrobiological science, 2001, 45～48.
[12] 洪华珠 彭建新．一株高水平表达重组蛋白昆虫细胞系的建立[J]．昆虫学报，2001，44(3)：276～280．
[13] 李晓锋 齐义鹏 朱应等．杆状病毒凋亡抑制基因的稳定转化及抗凋亡昆虫细胞系的构建[J]．昆虫学报，1999，42(2)：120～125．
[14] Cle m R J, Marcus F, Miller L K. Prevention of apoptosis by a baculovirus gene during infection of insect cells[J]. Science, 1991, 254:1388～1390.
[15] Shizuo G K, Majimak and Maedas. Identification and characterization of p35 gene of Bomyx mori nuclear polyhedrosis virus can prevents virus-induced apoptosis[J]. J. Virol, 1993, 67(1): 455～463.
[16] Norman E C, Cle m R J, Miller L K. An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif[J]. J. Virol, 1993, 67 (4): 2168～2174.
[17] Bimbaum M J. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypetide with Cys/His sequence motifs[J]. J. Virol, 1994, 68: 2521～2528.
[18] Palli S R, Caputo G F, Sohi S S. et al. CfMNPV blocks AcMNPV -induced apoptosis in a continuous midgut cell line[J]. J. Virol, 1996, 222:201～213.
[19] Gong M, Guarino L A. Expression of the 39k promotor of AcNPV is increased by the apoptotic suppressor p35[J]. J.Virol, 1994, 204: 38～44.
[20] Bernard A R,et al. Recombinant protein expression in a Drosophila cell line:comparison with the baculovirus system[J]. Cytotechnology, 1994, 15(13): 139～144.
[21] Kovach M J,Carlson J O,Beaty B J.A. Drosophila metallothionein promoter is inducible in mosquito cells[J]. Insect Mol.Biol., 1992, 1: 37～43.
[22] 黎路林，王峋章，陈曲侯．已经鉴定的杆状病毒基因及其功能[J]．病毒学报，1997，13(1)：88．
[23] Ayres MD, Howard SC, Kuzio J, et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus[J]. Virology, 1994, 202(2): 586～605.
[24] Theilmann DA, Chantler JK, Stweart S, et al. Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions[J]. Virology, 1996, 218(1): 148～158.
[25] Luckow VA, Summers MD. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors[J]. Virology, 1989, 170(1): 31～39.
[26] 侯云德．分子病毒学[M]．北京：学苑出版社．1990，72-86．
[27] 杜瑞娟．杆状病毒表达系统的发展[J]．生物技术通讯，1999，10(4)：310～313．
[28] David RO et al. Baculovirus expression vector[M]. New York: W-H Freeman and Company, 1992, 9.
[29] Todd J W, Passarelli AL, Lu A, et al. Factors regulating baculovirus late and very late gene expression in transient-expression assays[J]. J. Virol, 1996, 70(4): 2307～2317.
[30] Sriram S, Palhan VB, Gopinathan KP. Heterologous promoter recognition leading to higlrlevel expression of cloned foreign genes in Bombyx mori cell lines and larvae[J]. Gene, 1997, 190(1): 181～189.
[31] Smith G E, Summers M D, Fraser M J. Production of human beta interferon in insect cells infected with a baculovirus expression vector[J]. Molecular and Cellular Biology, 1983, 3: 2156～2165.
[32] Maeda S, Kawai T, Obinata M, et al. Characteristics of human interferonalpha produced by a gene transferred by a baculovirus vector in the silkworm, Bombyx mori[C]. Proc Jpn Acad, 1984, 60: 423～426.
[33] 吕鸿声．昆虫病毒分子生物学[M]．北京：中国农业科技出版社，1998，520-529，554-580．
[34] 骆学农 才学鹏．杆状病毒表达载体的研究及应用．中国兽医科技，2001，31(7)：13～16．
[35] Sisk-WP, Bradley JD, Leipold RJ, et al. High-level expression and purification of secreted
forms of herpes simplex Virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells[J]. J.Virol.1994, Feb, 68(2): 766～775.
[36] Carbonell LF, Klowden MJ, Miller LK. Baculovirus-mediated expression of bacterial genesindipteran and mammalian cells[J]. J.Virol, 1985, 56: 153～160.
[37] Hu SL, Kosowski SG, Schaaf KF. Expression of envelope glycoproteins of human immunodefciency virus by an insect Virus[J]. J.Virol, 1987, 61: 3617～3620.
[38] Inumaru S, Roy P. Production and characterization of the neutralization antigen Vp2 of bluetongue virus serotype 10 using a baculovirus expression vector[J]. Virology, 1987, 157: 472～479.
[39] Inumarus S, Ghiasi H, Roy P.. Expression of bluetongue virus group specific antigen VP3 in insect cells by a baculovirus vector: its use for the detection of bluetongue virus antibodies[J] , J Gen Virol, 1987, 68: 1627～1635.
[40] Chai H, Vasudevan SG, Porten AG, et al. Glycosylation and high-level secretion of human tumour necrosis factor-beta in recombinant baculovirus-infected insect cells[J]. Biotechnol Appl Biochem, 1993, 18(3): 259～273.
[41] Dai X, Hajos JP, Joosten NN, et al. Isolation of a Spodoptera exigua baculovirus recombinant with a 10. 6 kbp genome deletion that retains biological activity[J]. J Gen Virol, 2000, 81(10): 2545～2554.
[42] Marumoto Y, Sato Y, fujiwara H. et al. Hyper production of polyheardin IGF Y fusion protein in silkworm laryae infected with recombinant Bombyx mori nuclear polyhedrosis virus[J]. J Gen virol, 1998, 7(68): 2599～2606.
[43] Hirowatari Y, Hijikata M, Tanji Y, et al. Two proteinase activities in HCV Polypeptide expressed in insect cells using baculovirus vector[J]. Arch-Virol, 1993.
[44] Ailor E, Betenbaugh MJ.Modifying secretion and post- translational processing in insect cells[J]. Curr Opin Biotechnol., 1999,10 (2): 142～145.
[45] 朱帮福，卢兹凡．昆虫杆状病毒表达系统研究进展[J]．细胞与分子免疫学杂志，2002，18(6)：681～684．
[46] 朱立平．蛋白质糖基化与B细胞免疫[J]．上海免疫学杂志，2001，21(4)：193-194．
[47] 朱江 吴祥甫．昆虫杆状病毒表达系统研究进展及其应用展望[J]．蚕业科学，2003，29(2)：114～118．
[48] 张应玖 金宁一 金善荣．用细菌／杆状病毒系统在昆虫细胞中表达HIV-2外膜糖蛋白gp105和跨膜糖蛋白gp36[J]．免疫学杂志，2001 17(1)：13～16．
[49] 许强 王克夷．异源表达系统中蛋白质糖基化[J]．生物化学与生物物理学报，1999，31(2)：111～115．
[50] James DC. Freedman RB. Hoare M et al, N-gycosylation of recombinant huaman interferon-γ produced In different animal expression systems[J]. Bio/Tech-nology, 1995, 13: 592～596.
[51] Gonah O W, Freedman RB, Jenkins N, Patel K, Rooney B C. Isolation and characterization
of an insect-cell line able to perform complex N-linked glycosylation on recombinant proteins[J]. Bio/Technology, 1996, 14: 197～202.
[52] Joshi L, Davis TR, MattuTS, et al Influence of baculovirus-host human cell interaction on complex N-linked glycosylation of a recombinant human protein[J]. Biotechnol Prog, 2000, 16(4): 650～656.
[53] Matsuura Y, Possee RD, Overton HA, DHL. Baculovirus expression vectors therequirements for high level expression of proteins Including glycoprotiens [J]. J Gen Virol, 1987, 68: 1233～1250.
[54] Jeang KT, Glam CZ, Nerenberg M, et al. Abundant synthesis of functional human T—cell leukemia Virus typel p40 protein in eucaryoticc ellsby usinga baculovirus expression vector[J].J Virol, 1987, 61: 708～713.
[55] Sandoval C,Curtis H,Congote LF.Enhanced proliferative effects and alpha(1)-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351[J]. Protein Eng, 2002, 15(5): 413～418.
[56] Prehaud C,Takehara K. Immunognic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors [J]. Virology, 1989, 173: 390～399
[57] 周压竞 张志方 张元兴等．昆虫细胞培养研究进展[J] ，蚕业科学 2000，26(增刊)：74-78．
[58] Barbanti Brodano G, Martini F, DeMattei M et al. BK and JC human polyomavirus and simain virus 40: natural history of infection in humans expermiental oncogenicity and association with human tumor[C]. Adv Virus Res, 1998, 50: 69～99.
[59] 刘馨．SV40(Simain virus 40)的分子生物学研究进展[J]．国外医学病毒学分册，2002，9(3)：65～68．
[60] Schlze-Garg C, et al. Ontogene, 2000, 8: 1020-1027.
[61] 解慧琪 杨志明 屈艺．SV40与细胞永生化[J]．中国修复重建外科杂志，2000，14(3)：170～174．
[62] Reilly C F, Rat vascular smooth muscle cells immortalized with SV40 large T antigen possess defined smooth muscle cell characteristics including growth inhibition by heparin[J]. J Cell Physiol. 1990, 142 (2): 342
[63] Su RT, Bradford L. Keratin synthesis in SV40 transformed human keratinocytes after supertransfection with Harvey murine sarcoma viral DNA[J]. Exp Cell Res 1993,205(2): 403.
[64] Mqud C, Caghowx P, Durand C, et al. Establishment and characterization of cell line LSV5 that retains the altered adhesive properties of human junctional epidemolysis bullosa keratinocyte[J]. Exp Cell Res.1996, 224(2): 279.
[65] Webber M M, Bello D, Kleinman H K, et al. Prostate specific antigen and androgen receptor induction and characterization of an immortalized adult human prostatic epithelial cell line[J]. Carcinogenesis.1996,17 (8): 1641.
[66] Racusen LC, Monteil C, Sgrinoli A, et al. Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization response to inducers and comparison with established cell lines[J]. J Lab Clin Med, 1997, 129(3): 318.
[67] Houton A,Oyajobi B, Foster G, et al. Immortalization of human marrow stromal cells by retroviral transduction with a temperature sensitive oncogene: Identification of bipotential precursor cells capable of directed differentiation to either an osteoblast or adipocyte phenotype[J]. Bone, 1998, 22(10): 7.
[68] Wang P.,Granados,R.R.,Shuler, M.L. Studies on serum-free culture of insect cells for virus propagation and recombinant protein production[J]. J. Invertebr. Pathol., 59: 46-53.
[69] Hayflick L, Theory of population increase by subcultivation[A]. Kruse P.F., and Patterson., M K. In "Tissue Culture Methods and Application" [C] , Academic Press, NY 1973,222-223.
[70] Jarvis, D.L. Baculovirus expression vectors[A]. In Miller, L K (ed.), The bacutoviruses[C]. Plenum press, New York, 1997,389-431. New York, NY, Plenum Press 447.
[71] Granados, R.R., Guoxun.L., Derksen A.C.G.,&Mckenna K.A. A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni singly enveloped nuclear polyhedrosis virus[J]. J. Invertebr. Pathol., 1994, 64: 260～266.
[72] Clemm, D.L., Scale-up of protein production in a stirred bioreactor[A]. In D.R.O'Relilly, L.K.Miller&V.A.Luckow, Eds. Baculovirus Expression vectors, a Laboratory Manual[C]. New York, NY, W.H.Freeman and Company. 1992,447.
[73] Wong, K.T.K., Peter, C.H., Greenfield, P.F., Reid, S. &Nielson, L.K. Low multiplicity infection of insect cells with a recombination baculovirus: the cell yield concept[J]. Biotechnol. Bioeng., 1996.49: 659～666.
[74] Donaldson M.S.& Shuler M.L. Effects of long-term passaging of BTI-Tn-5B1-4 insect cells on growth and recombinant protein production[J]. Biotechnol.Prog., 1998, 14: 543～547.
[75] Wickham T J, Davis,T, Granados RR, Shuler M L, &Wood,H A. Screening of insect cell lines for the production of recombinant and infectious virus in the baculovirus expression system[J]. Biotechnol Prog. 1992, 8: 391～396.
[76] Yang K,Mu X S, Hayes R L, et al. DC--Cholliposome--mediated gene transfer in rat spinal cord[J]. Neuroreport. 1997, 8 (9): 2355.
[77] 卢圣栋．外源基因在真核细胞中的表达[M]．现代分子生物学实验技术．北京：高等教育出版社，1993，389～403．
[78] 萨姆布鲁克 J，弗甲奇 EF，曼尼阿蒂斯 T，著．分子克降实验指南，第2版北京：科学出版社，1992，765～780．
[79] Clem R J, Miner L K, Apoptosis reduces both the in vitro replication and in vivo infectivity of a baculovirus[J]. J Virol 1993, 67(7): 3730～3738.
[80] Palli S R,Caputo G F, Sohi S S et al. CfMNPV blocks AcMNPV-induced apoptosis in a
continuous miggut cell line[J]. J Virol., 1996, 222: 201～203.
[81] Nor Chjanovsky, Eduard Gershburg. The wild-type AcNPV induces apoptosis of Spodoptera littoralis cells[J]. Virol 1995,209: 519～525.
[82] Granados R R,Mc Kenna K A. Insect cell culture methods and their use in virus research[A]. In: Shuler M L,Wood H A. Granados R R et al. eds. Baculovirus expression systems and Biopesticides[C]. New York. Wiley Liss, 1995, 13～39.
[83] Miller L K. Baculovirus expression vectors[C]. Annu Rev Microbiol.1988. 42: 177～199.
[84] Davis T R,Trotter K M, Granados R R et al. Baculovirus expression of alkaline phosphatase as a reporter gene for evolution of production, glycosylation and secretion[J]. Biotechnology, 1992, 10: 1148～1150.
[85] Davis T R, Wichham T J,Mckenna KA et al. Comparative recombinant protein production of eight insect cell lines[J]. In vitro cell. Dev.Bio.,1993, 29: 388～390.
[86] Davidof A N, Nendelow B V. Identifications of endonuclease activity in HIV-1 gp120 preparation systems[J]. Biotechiques. 1997, 23(2): 296～299.
[87] Lian M.Guttieri M, Lundkvist A,et al. Baculovirus expression of human G2-specific,neutralizing IgG monoclonal antibody to Puumala virus [J]. Virology, 1997, 235 (2):252～260.
[88] Shinkai A,Shinodac K,Morishita Y et al. High level expression alpa -1,3-fucosyltranferase in Baculovirus-infected insect cells[J]. Protein Express. Purif., 1997, 10(3): 397～385.
[89] Haining R L,Bunter A P, Sadeque A J,et al. Baculovirus-mediated expression and purification of human FMO3:catalytic.Immunochemical,and structural characterization[J]. Drug Metab.Dispos., 1997, 25(7): 790～797.
[90] King G A, Faulkner P, Daugulis A J et al. Insect cell cultivation and baculovirus gene expression in serum-free medium.199 ACS National meeting, Boinsect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system[J]. Biotechnol.Prog., 1990, 8: 391～396.
[91] Hink W F, Thomsen D R, Daidson D J,et al. Expression of three recombinant proteins using baculovirus vector in 23 insect cell lines[J].Biotechnol. Prog., 1991, 7: 9～14.
[92] Ogonah O, Shuler M L,Granados R R. Protein production (β-galactosidase) from a baculovirus vector in Spodoptera frugiperda and Trichoplusia ni cells in suspension culture[J]. Biotechnol.,Lett., 1991, 13: 265～270.
[93] Wichham T J, Davis T, Granados R R. et al. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system[J]. Biotechnol.Prog., 1992, 8: 391～396.
[94] Bibeiro B M, Crook N E, Expression of full-length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp.kurstaaki in a baculovirus and pathogenicity of the recombinant virus [J]. J. Invertebr. Pathol., 1993, 62: 121～130.
[95] Chen Q, Mctosh A H, Yu Z et al. The replication of Autographa californica baculovirus (AcMNPV) in two lepidopteran cell lines grown in serum-free media[J]. J. Invertebr. Pathol., 1993, 62: 216～219.
[96] Yamada, K I.,Sherman,K.E.,Maramorosch,K.Serial passage of Heliothis zea single embedded nuclear polyhedrosis virus in a homologous cell line [J]. J.Invertebr. Patho.,1982, 39: 182～191.
[97] 小池胜 通过大量培养昆虫细胞生长天敌病毒[J]．国外农学：植物保护，1992，5(3)：26～29．
[98] 裘卫，朱国凯．苜蓿丫纹夜蛾杆状病毒在四个异源细胞中连续传代复制[J]．病毒学报，1987，3(3)：259～262．
[99] 田波，龚祖埙．病毒与农业[M]．科学出版社，1986．