表达鸡马立克氏病病毒糖蛋白B基因及共表达糖蛋白B和鸡γ干扰素基因的重组鸡痘病毒的构建及其免疫保护作用
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摘要
鸡马立克氏病（MD）是由马立克氏病病毒（MDV）引起的一种淋巴组织增生
性肿瘤疾病，常因造成免疫抑制和大批死亡而给养禽业带来巨大的经济损失。虽
然常规疫苗在控制MD的发生中起到非常重要的作用，但也存在难以克服的缺点，
常规的HVT冻干疫苗使用方便而免疫效力欠佳，常规的液氮冷冻疫苗保护效力高
而保存和运输不方便。为了能研制新一代安全、高效、使用方便的基因工程疫苗，
本研究对鸡痘病毒的复制非必需片段进行优选，对人工合成启动子进行组合筛选，
以优选的复制非必需片段和强启动子构建了高效表达MDV gB基因的重组鸡痘病
毒及共表达MDV gB和γ干扰素基因的重组鸡痘病毒（rFPV），并在不同品种鸡中
测定了rFPV的免疫效力。现从五个方面将本论文进行的研究概述如下：
一、鸡痘病毒复制非必需片段的优选
pSV-Gal经HindⅢ和BamHI双酶切回收β-半乳糖苷酶基因（LacZ）片段，插
入到BamHI单酶切的质粒pp18中P7.5下游，构建成含P7.5-LacZ基因盒的pB7.5，
以HindⅢ和XbaI双酶切pB7.5，回收P7.5-LacZ基因盒，插入到3个含鸡痘病毒
复制非必需片段的质粒pFPV18、pFPV7s、pFPV9s的EcoRV位点构建成转移载体
pFB18、pFB7s和pFB9s，以转移载体和脂质体共转染已感染FPV中国疫苗株282E4
的鸡胚成纤维细胞（CEF），经多次蓝斑筛选和纯化，得到稳定生长的重组病毒；
将纯化后的重组鸡痘病毒感染次代CEF，于感染后72小时收获细胞，制备细胞提
取物，检测提取物中β-半乳糖苷酶的活性。通过比较活性的高低及片段长短的优
势，我们选取了pFPV7s作为构建高效表达载体的复制非必需片段。经序列测定，
该片段由2909个核苷酸组成，是新鉴定出的FPV复制非必需片段。
－d－ler。es
n ie州大学博士学位论文
二、鸡痘病毒强启动子的合成和筛选
根据痘苗病毒天然启动子P7．5的结构，考虑核昔酸的种类、数目、分布与启
动效率之间的关系，人工合成一条寡核昔酸，经磷酸化，变性退火后各形成一个
早期G）和一个晚期启动子（L），其首尾可相互连接；用 T4 DNA连接酶使早、
晚期启动子自身连接或混合连接，将不同启动子的组合克隆到pUC t体中，通
过原位杂交筛选，酶切、电泳及测序来确定启动子数目，获得了IO个较有代表性
的启动子组合P81．10：PSI（L）、PSZ（LL）、PS3（LLLL）、Ps4（EE）、PSS（LE）、
Ps6（LELE）、Ps7（LLEE）、Pss（LLEEEE）、Psg（LLES）和 Pslo（LLE18）。在
质粒Psllo的不同启动子下游插入LacZ基因后，用BamHI和Hindlll双酶切回收
PS－LacZ基因插人到含鸡痘病毒复制非必需片段的质粒pFPV7S形成转移载体
pFBSllo，脂质体转染，蓝斑筛选和纯化得到重组病毒OFPVSllo）；以含P7．5－eZ
基因的重组病毒为对照，含合成启动子的重组病毒于感染CEF后10、24、36、48、
72小时收获细胞，制备细胞提取物，检测p－半乳糖昔酶的活性。通过活性强弱的比
较，筛选出最强的启动子组合LLEE，其表达活性为P7．5的3．5倍。和其表达效率
相近的启动子组合为LLLL。我们选用LLEE来构建高效表达载体。
三～高效表达MDVgB＆因的重组鸡痘病毒的构建
人工合成2＆寡聚核昔酸，形成一个多克隆位点，与EcoRV＄切的pFPV7s
相连形成含多克隆位点的yV7S；将pG18用BamHI酶切，回收PlllncL基因盒
插入到含强启动子LLEE的PS的EcoAI位点构建pOS，使LLEE与Pll－eZ处于
相对方向；再将 pGS中的 PSLacZPll基因盒插入到 FPV7S中构建成插入载体
PFGSll 最后将 CVI988 gB基因插入到 pFGSll中的 PS下游构建成转移载体
pFGBS，利用 PS来调控 MDV gB的表达；通过酶切和 SOUthern blot鉴定后，转染
已感染 FPV 282E4的 CEF，经蓝斑筛选和纯化得到重组病毒rFPVPS－gB，以抗
MDV gb单抗作间接免疫荧光试验可见感染重组病毒的CEF的细胞膜和细胞浆有
特异性的黄绿色的荧光，与P7．5调控MDV gB基因表达的重组病毒dpVI7．5－gB
相比，d7T，v－PS伊的gB基因的表达量是后者3倍以上。证明了优选的复制非必需
片段可用于构建稳定的鸡痘病毒载体，筛选的强启动子可提高目的基因的表达量。
四、共表达MDVgh和可干扰素基因（WN）的重组鸡痘病毒的构建
彭大新：表达 MDV gB基因及共表达 gB和鸡 Y干扰素基因的重组鸡痘病毒的构建及其免疫保护作用 f
将含 PE／LJFN基因盒的质粒用 EcoM酶切补平后插入到 pFGBS的 Smal位点，
通过酶切鉴定和斑点杂交获得了PEIJFN与PS唱B同向和相对的转移载体
pFGBSll和pFGBSIZ。选择pFGBSIZ经脂质体转染，蓝斑筛选纯化得到稳定的重
组病毒rFPVPS唱B工FNJ。以间接兔疫荧光法证实，LLEE调控的MDV gB可正
Doctor candidate: PENG DAXIN
Advisor: Prof LW XTUFAN
In order to develop Marck抯 disease (MD) genetic engineering vaccines which are safe, convenient for use and with high protective efficacy, We designed and completed a series of experiments to reach the goal. Novel nonessential fragments of fowlpox virus (FPV) were identified, and the best one was selected by comparison to construct transfer vector, synthetic promoter, which is 3.5 times stronger than the P7.5 promoter of vaccinia virus was constructed and used for vector construction; recombinant FPVs expressing MDV gB or coexpressing MDV gB and type II interferon were constructed and assayed; recombinant viruses were evaluated for their protective efficacy in vaccination trials with specific-pathogen-free(SPF) and commercial chickens.
1. Optimization of newly identified nonessential fragments of Fowlpox virus for vector construction
The LacZ gene from plasmid pSV-Gal was inserted into the downstream of P7.5 to form plasmid pB7.5. For construction of Fowlpox virus (FPV) transfer vectors, the P7.5-LacZ cassettes from pB7.5 was removed and inserted into nonessential regions of FPV in plasmid pFPV18, pFPV7s and pFPV9s to form pFBI8, pFB7s and pFB9s. These transfer vectors were lransfected with DOS PER liposome into chicken embryo fibroblast(CEF) monolayers pre-infected with Chinese vaccine strain 282E4 of FPV. Recombinant viruses were purified by selection of plaques expressing ~ -galactosidase in secondary CEF cultures overlaid with agar containing X-Gal. The viruses were propagated and titrated by the plaque assay in CEF. The level of the ~ -galactosidase expressed by rFPVs in CEF extraction was assayed and compared at 72 hours post infection. Results suggested that the pFPV7s was the best nonessential fragment suitable for foreign gene insertion, in terms of the stability and intensity of foreign gene expression in recombinant viruses.
MDV gB ~LU~ gB ~UX~ Y ~
2. Synthesizing and selecting stronger promoters for transfer vector construction
Four olignucleotides were synthesized according to the essential structure of the vaccina virus promoter P7.5 and were used to generate an early (E) and a late(L) promoter. Ten artificial promoters designated Psl-1O were cloned into plasmid pUC18 by using different numbers and combinations of early and late promoters in tandem, selected by colony hybridization and identified by restriction endnuclease cleavage patterns and sequence analysis. The LacZ gene was removed and inserted into the downstream of different promoters, then the Ps-LacZ cassettes were inserted into pFPV7s respectively to form transfer vectors pFBS1-10. rFPVs containing different promoters were constructed through the method of homologous recombination. Extracts of CEF infected with rFPVs were prepared at 10, 24, 36, 48 and 72 hours post infection. The quantity and level of P -galactosidase expression were assayed by colorimetry in the presence or absence of cytosine arabinoside. Results showed that the LLEE combination was the strongest synthetic promoter which drove expression of P -galactosidase 3.5 times higher than that of P7.5.
3. Construction of recombinant fowlpox (rFPV) virus expressing MDV gB under the control of the strong promoter
FPV insertion vector was constructed with promoter LLEE and P11 -LacZ marker gene cassette in opposite direction which flanked with nonessential fragment from FPV7s. MDV CVI 988 gB was inserted into the downstream of LLEE to form transfer vector pFGBS. Recombinant fowlpox viruses were selected by blue plaque and the expression of MDV gB by rFPV was confirmed by indirection immunofluorescence assay (IFA). Expression level of MDV gB driven by promoter LLEE in rFPV was 3 times higher than that of MDV gB driven by viccina virus P7.5 promoter.
4. Construction of recombinant fowlpox virus (rFPV) coexpressing MDV gB and type II interferon
Recombinant fowlpox viruses coexpressing MDV gB and chicken type II interferon(IFN-II) gene was constructed by using diff
性肿瘤疾病，常因造成免疫抑制和大批死亡而给养禽业带来巨大的经济损失。虽
然常规疫苗在控制MD的发生中起到非常重要的作用，但也存在难以克服的缺点，
常规的HVT冻干疫苗使用方便而免疫效力欠佳，常规的液氮冷冻疫苗保护效力高
而保存和运输不方便。为了能研制新一代安全、高效、使用方便的基因工程疫苗，
本研究对鸡痘病毒的复制非必需片段进行优选，对人工合成启动子进行组合筛选，
以优选的复制非必需片段和强启动子构建了高效表达MDV gB基因的重组鸡痘病
毒及共表达MDV gB和γ干扰素基因的重组鸡痘病毒（rFPV），并在不同品种鸡中
测定了rFPV的免疫效力。现从五个方面将本论文进行的研究概述如下：
一、鸡痘病毒复制非必需片段的优选
pSV-Gal经HindⅢ和BamHI双酶切回收β-半乳糖苷酶基因（LacZ）片段，插
入到BamHI单酶切的质粒pp18中P7.5下游，构建成含P7.5-LacZ基因盒的pB7.5，
以HindⅢ和XbaI双酶切pB7.5，回收P7.5-LacZ基因盒，插入到3个含鸡痘病毒
复制非必需片段的质粒pFPV18、pFPV7s、pFPV9s的EcoRV位点构建成转移载体
pFB18、pFB7s和pFB9s，以转移载体和脂质体共转染已感染FPV中国疫苗株282E4
的鸡胚成纤维细胞（CEF），经多次蓝斑筛选和纯化，得到稳定生长的重组病毒；
将纯化后的重组鸡痘病毒感染次代CEF，于感染后72小时收获细胞，制备细胞提
取物，检测提取物中β-半乳糖苷酶的活性。通过比较活性的高低及片段长短的优
势，我们选取了pFPV7s作为构建高效表达载体的复制非必需片段。经序列测定，
该片段由2909个核苷酸组成，是新鉴定出的FPV复制非必需片段。
－d－ler。es
n ie州大学博士学位论文
二、鸡痘病毒强启动子的合成和筛选
根据痘苗病毒天然启动子P7．5的结构，考虑核昔酸的种类、数目、分布与启
动效率之间的关系，人工合成一条寡核昔酸，经磷酸化，变性退火后各形成一个
早期G）和一个晚期启动子（L），其首尾可相互连接；用 T4 DNA连接酶使早、
晚期启动子自身连接或混合连接，将不同启动子的组合克隆到pUC t体中，通
过原位杂交筛选，酶切、电泳及测序来确定启动子数目，获得了IO个较有代表性
的启动子组合P81．10：PSI（L）、PSZ（LL）、PS3（LLLL）、Ps4（EE）、PSS（LE）、
Ps6（LELE）、Ps7（LLEE）、Pss（LLEEEE）、Psg（LLES）和 Pslo（LLE18）。在
质粒Psllo的不同启动子下游插入LacZ基因后，用BamHI和Hindlll双酶切回收
PS－LacZ基因插人到含鸡痘病毒复制非必需片段的质粒pFPV7S形成转移载体
pFBSllo，脂质体转染，蓝斑筛选和纯化得到重组病毒OFPVSllo）；以含P7．5－eZ
基因的重组病毒为对照，含合成启动子的重组病毒于感染CEF后10、24、36、48、
72小时收获细胞，制备细胞提取物，检测p－半乳糖昔酶的活性。通过活性强弱的比
较，筛选出最强的启动子组合LLEE，其表达活性为P7．5的3．5倍。和其表达效率
相近的启动子组合为LLLL。我们选用LLEE来构建高效表达载体。
三～高效表达MDVgB＆因的重组鸡痘病毒的构建
人工合成2＆寡聚核昔酸，形成一个多克隆位点，与EcoRV＄切的pFPV7s
相连形成含多克隆位点的yV7S；将pG18用BamHI酶切，回收PlllncL基因盒
插入到含强启动子LLEE的PS的EcoAI位点构建pOS，使LLEE与Pll－eZ处于
相对方向；再将 pGS中的 PSLacZPll基因盒插入到 FPV7S中构建成插入载体
PFGSll 最后将 CVI988 gB基因插入到 pFGSll中的 PS下游构建成转移载体
pFGBS，利用 PS来调控 MDV gB的表达；通过酶切和 SOUthern blot鉴定后，转染
已感染 FPV 282E4的 CEF，经蓝斑筛选和纯化得到重组病毒rFPVPS－gB，以抗
MDV gb单抗作间接免疫荧光试验可见感染重组病毒的CEF的细胞膜和细胞浆有
特异性的黄绿色的荧光，与P7．5调控MDV gB基因表达的重组病毒dpVI7．5－gB
相比，d7T，v－PS伊的gB基因的表达量是后者3倍以上。证明了优选的复制非必需
片段可用于构建稳定的鸡痘病毒载体，筛选的强启动子可提高目的基因的表达量。
四、共表达MDVgh和可干扰素基因（WN）的重组鸡痘病毒的构建
彭大新：表达 MDV gB基因及共表达 gB和鸡 Y干扰素基因的重组鸡痘病毒的构建及其免疫保护作用 f
将含 PE／LJFN基因盒的质粒用 EcoM酶切补平后插入到 pFGBS的 Smal位点，
通过酶切鉴定和斑点杂交获得了PEIJFN与PS唱B同向和相对的转移载体
pFGBSll和pFGBSIZ。选择pFGBSIZ经脂质体转染，蓝斑筛选纯化得到稳定的重
组病毒rFPVPS唱B工FNJ。以间接兔疫荧光法证实，LLEE调控的MDV gB可正
Doctor candidate: PENG DAXIN
Advisor: Prof LW XTUFAN
In order to develop Marck抯 disease (MD) genetic engineering vaccines which are safe, convenient for use and with high protective efficacy, We designed and completed a series of experiments to reach the goal. Novel nonessential fragments of fowlpox virus (FPV) were identified, and the best one was selected by comparison to construct transfer vector, synthetic promoter, which is 3.5 times stronger than the P7.5 promoter of vaccinia virus was constructed and used for vector construction; recombinant FPVs expressing MDV gB or coexpressing MDV gB and type II interferon were constructed and assayed; recombinant viruses were evaluated for their protective efficacy in vaccination trials with specific-pathogen-free(SPF) and commercial chickens.
1. Optimization of newly identified nonessential fragments of Fowlpox virus for vector construction
The LacZ gene from plasmid pSV-Gal was inserted into the downstream of P7.5 to form plasmid pB7.5. For construction of Fowlpox virus (FPV) transfer vectors, the P7.5-LacZ cassettes from pB7.5 was removed and inserted into nonessential regions of FPV in plasmid pFPV18, pFPV7s and pFPV9s to form pFBI8, pFB7s and pFB9s. These transfer vectors were lransfected with DOS PER liposome into chicken embryo fibroblast(CEF) monolayers pre-infected with Chinese vaccine strain 282E4 of FPV. Recombinant viruses were purified by selection of plaques expressing ~ -galactosidase in secondary CEF cultures overlaid with agar containing X-Gal. The viruses were propagated and titrated by the plaque assay in CEF. The level of the ~ -galactosidase expressed by rFPVs in CEF extraction was assayed and compared at 72 hours post infection. Results suggested that the pFPV7s was the best nonessential fragment suitable for foreign gene insertion, in terms of the stability and intensity of foreign gene expression in recombinant viruses.
MDV gB ~LU~ gB ~UX~ Y ~
2. Synthesizing and selecting stronger promoters for transfer vector construction
Four olignucleotides were synthesized according to the essential structure of the vaccina virus promoter P7.5 and were used to generate an early (E) and a late(L) promoter. Ten artificial promoters designated Psl-1O were cloned into plasmid pUC18 by using different numbers and combinations of early and late promoters in tandem, selected by colony hybridization and identified by restriction endnuclease cleavage patterns and sequence analysis. The LacZ gene was removed and inserted into the downstream of different promoters, then the Ps-LacZ cassettes were inserted into pFPV7s respectively to form transfer vectors pFBS1-10. rFPVs containing different promoters were constructed through the method of homologous recombination. Extracts of CEF infected with rFPVs were prepared at 10, 24, 36, 48 and 72 hours post infection. The quantity and level of P -galactosidase expression were assayed by colorimetry in the presence or absence of cytosine arabinoside. Results showed that the LLEE combination was the strongest synthetic promoter which drove expression of P -galactosidase 3.5 times higher than that of P7.5.
3. Construction of recombinant fowlpox (rFPV) virus expressing MDV gB under the control of the strong promoter
FPV insertion vector was constructed with promoter LLEE and P11 -LacZ marker gene cassette in opposite direction which flanked with nonessential fragment from FPV7s. MDV CVI 988 gB was inserted into the downstream of LLEE to form transfer vector pFGBS. Recombinant fowlpox viruses were selected by blue plaque and the expression of MDV gB by rFPV was confirmed by indirection immunofluorescence assay (IFA). Expression level of MDV gB driven by promoter LLEE in rFPV was 3 times higher than that of MDV gB driven by viccina virus P7.5 promoter.
4. Construction of recombinant fowlpox virus (rFPV) coexpressing MDV gB and type II interferon
Recombinant fowlpox viruses coexpressing MDV gB and chicken type II interferon(IFN-II) gene was constructed by using diff
引文
1 Omar A K, Schat K A, Lee L F et al. Cytotoxic T lymphocyte respone in chickens immunized with a recombinant fowlpox virus expressing Marek's disease herpesvirus glycoprotein B. Vet Immunopathol, 1998, 62(1):73-82.
2 Baxby D and Paolett L E. Potential use of none-replicating vectors as recombinant vaccines. Vaccines, 1992,10 (1):8-9.
3 Nazerian K, Witter R L, Lee L F et al. Protection and synergism by recombinant fowlpox vaccines expressing genes from Marek's disease vires. Avian Dis, 1996, 40(2):368-376.
4 Taylor J, Weinberg R. Kawaokay et al. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine, 1988, 6:504-508.
5 Ogawa R. Ynagida N Saekis et al. Recombinant fowlpox virus inducing protective immunity against Newcastle disease and fowlpox virus. Vaccine, 1990, 8:486-489.
6 彭大新，王志亮，刘秀梵等．鸡痘病毒基因组DNA复制非必需片段的鉴定．江苏农学院学报，1995，16 (4)：13-17．
7 J．萨姆布克等主编，金冬雁等译．分子克隆实验指南(第二版)．北京：科学出版社，1993：804-808．
8 金宁一，殷震，舟桥真一等．痘苗病毒高效表达载体的构建研究．中国兽医学报，1994，14 (1)：5-13．
9 Bernard P. A practical guide to molecular cloning. 2nd. USA:John Wiley & Sons Inc. 1988, 422-427.
10 Boyle D B Coupar B E. Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia vires. J. Gen Virol: 1986, 67(Pt8): 1591-1600.
11 Boursnell M, Green P F, Campbell J I, et al. Insention of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant. J Gen Virol, 1990, 1:621-628.
12 Boyle D B amd Coupar B E H. Coustruction of recombinant fowlpox virus as vectors for poultry vaccines. Virus research, 1988, 10:343-356.
13 Davian A J, Moss B. Structure of vaccinia virus early promoters. J. Mol. Biol. 1989, 210:749-769.
14 Davian A J, Moss B. Structure of vaccinia virus late promoters. J. Mol. Biol. 1989,210:771-784．
15 Yanagida N, Ogawa R, Li Y et al. Recombinant fowlpox viruses expressing the glycoprotein B homology and the pp38 of Marek's disease virus. J. Virol, 1992, 66:1402-1408．
16 Boyle D B. Quantitative assessment of poxvirus promoters in fowlpox and vaccinia virus recombinants. Virus Genes, 1992, 6(3) :281-290．
17 Coupar B E H, Andrew M E, Both G et al. Temporal regulation of influenza hemagglutinin expression in vaccnia virus recombinants and effects on the immune response. Eur. J. Immunol., 1986, 16:1479．
2 Baxby D and Paolett L E. Potential use of none-replicating vectors as recombinant vaccines. Vaccines, 1992,10 (1):8-9.
3 Nazerian K, Witter R L, Lee L F et al. Protection and synergism by recombinant fowlpox vaccines expressing genes from Marek's disease vires. Avian Dis, 1996, 40(2):368-376.
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