CN103255171B - 猪圆环病毒2型密码子优化的orf2基因的重组病毒样粒子 - Google Patents
猪圆环病毒2型密码子优化的orf2基因的重组病毒样粒子 Download PDFInfo
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Abstract
本发明涉及猪圆环病毒(PCV)2型密码子优化的ORF2基因重组杆状病毒病毒样粒子,属于生物制药领域。将所有PCV2 ORF2序列进行比对分析,得到人工合成的密码子优化的PCV2 ORF2序列,命名为YHsfORF2。将YHsfORF2克隆、转染,获得重组杆状病毒vFBHYHsfORF2。密码子优化的ORF2基因能够在重组病毒中高效表达,并能形成病毒样颗粒。本发明还涉及该重组病毒在疫苗方面的应用,动物免疫保护试验表明该重组病毒灭活疫苗,可刺激机体产生高效的免疫应答,有效抑制PCV2在动物体内的复制,对PCV2感染有较好的免疫预防保护作用。
Description
一、技术领域
本发明涉及猪圆环病毒2型密码子优化的ORF2基因的重组病毒样粒子,属于生物工程领域,具体地说,涉及猪圆环病毒ORF2密码子的优化、优化序列重组杆状病毒的获得、重组病毒的制备及基因工程疫苗,属于基因工程疫苗领域。
二、背景技术
猪圆环病毒病(PCVD)是由猪圆环病毒2型(PCV2)引起的猪传染性疾病,是猪的重要免疫抑制性疾病之一,该病已给全球养猪业造成了巨大的经济损失。众多学者认为,猪圆环病毒病会引起断奶后仔猪多系统衰弱综合征(PMWS)及猪皮炎肾病综合征(PDNS),同时还能引起母猪繁殖障碍、新生仔猪先天性震颤(CT)等疾病,也是猪呼吸道疾病综合征(PRDC)的原发病原之一。目前,国内外猪场普遍存在PCV2感染,我国自2000年报道PCV2感染的血清学证据以来,流行范围已波及全国,猪群平均阳性率最高高达60%,发病猪死亡率为10%-30%不等,较严重的猪场在爆发本病时死亡率高达40%,危害日益严重。
目前,如何有效防控PCVD已成为全球养猪业关注的焦点。疫苗预防接种是控制本病的最有效手段,国内外研究者也在猪圆环病毒病疫苗研制方面开展了较多工作。首先是全病毒灭活疫苗,目前国内外都有注册的灭活疫苗,但总体而言,灭活疫苗对机体刺激时间比较短,需多次接种,而且PCV2在细胞上增殖滴度低,其制备费用远远高于其他疾病的灭活疫苗;其次是亚单位疫苗,相对于其他疫苗,亚单位疫苗产生抗体更早,抗体水平稳定,能够有效阻止病毒的复制,保护力强,目前国外利用杆状病毒表达的Cap蛋白的亚单位疫苗已有注册,Fort M等研究也表明该疫苗使用剂量相对较少,免疫效果好,对欧洲及北美的PCV2控制发挥了巨大作用;再次是减毒活疫苗,Fenaux M等学者发现了PCV2的致弱位点,为减毒活疫苗的研制提供了方向,该疫苗虽然能够诱导很好的免疫应答,但长期使用存在反强的危险;另外也有学者对其活载体疫苗、核酸疫苗等进行研究,但都没有获得成品。众多研究表明,PCV2疫苗的研制方向应该是基因工程疫苗,尤其是亚单位疫苗。
目前国外勃林格殷格翰动物保健公司的PCV2疫苗(CircoFlex)已在我国上市,2010年国内由姜平等和刘长明等研发的猪圆环病毒2型灭活疫苗,圆健-SH株和LG株也相继上市。国外疫苗价格十分昂贵,国内灭活疫苗又基于细胞增殖病毒,因PCV2在细胞上增殖滴度低、生产活疫苗和灭活疫苗费时又费力。因而在PCV2流行逐年上升,流行范围逐步扩大,各成长阶段的猪感染严重、混合感染突出、给全国养猪业造成了巨大损失的情况下,如何研制出一种免疫原性好、可以高效生产、价格合适的新型基因工程可商品化疫苗已成为当务之急。
PCV2主要编码3个阅读框,ORF1编码与复制相关的蛋白即Rep蛋白;ORF2编码病毒的主要结构蛋白即Cap蛋白,是PCV2的主要免疫原;ORF3编码的蛋白与细胞凋亡有关。目前,由于Cap蛋白是主要的免疫原性蛋白,因而基于Cap蛋白的表达以研制成疫苗的研究成为热点。杆状病毒表达系统以其表达外源性蛋白产量高、抗原性好、产物易纯化等优点,在众多疫病的研究中得到广泛应用。目前研究表明,利用杆状病毒表达Cap蛋白能够自我组装成类病毒粒子,且刘长明、Fort M等研究表明,杆状病毒表达Cap蛋白具有很好的免疫原性。
目前,我国关于PCV2相关研究的报道,尤其是遗传变异情况的报道不断增多。有报道表明在中国,PCV2a可细分为2A、2D、2E亚型,PCV2b可细分为1A/1B、1C亚型,还可能存在既非PCV2a又非PCV2b的亚型PCV2c;温立斌等利用RFLP分析技术可将我国流行毒株分为9种基因型,即CHN-2A、CHN-2B、CHN-2C、CHN-2D、CHN-2E、CHN-2F、CHN-2G、CHN-2H和CHN-2I。可见我国PCV2存在基因多样性,因而根据单一的PCV2序列设计的疫苗无法对所有PCV2亚型提供很好的交叉保护。因而本研究将已知的PCV2基因进行系统进化树分析,取其一致序列(Consensus sequence),提高其交叉保护性。
免疫原基因的密码子优化已成为提高疫苗免疫保护效果的有效策略之一。这一策略已经在新城疫病毒、PRRSV、HBV等病毒的疫苗研究中被应用,试验结果表明其密码子优化可显著提高目的基因的表达,增强免疫效果。基于此,本研究将GenBank中所有PCV2序列比对分析,取ORF2基因的一致序列,通过人工合成的方法在不改变氨基酸序列的前提下,将密码子改造为昆虫细胞偏嗜的密码子,同时将相对于杆状病毒的稀有密码子进行优化和改造,提高表达效率。并将其作为免疫原基因, 插入杆状病毒表达系统,从而快速、大量获得表达产物。研究其免疫保护性,探讨其在抑制病毒和减少病毒血症方面的优势,从而研制出一种安全、高效的猪圆环病毒病毒样颗粒疫苗。
三、发明内容
技术问题
本发明的目的在于,提供一种通过基因比对的方法获得PCV2 ORF2 表达Cap蛋白氨基酸一致序列,提高交叉保护性。
本发明的另一个目的在于提供一种保持氨基酸不变的前提下,将密码子改造为杆状病毒偏嗜密码子,提高表达量。进行密码子优化时,优先选择昆虫细胞使用频率最高的密码子。根据具体情况(避免多个重复序列的出现等)作适当调整,若不能使用频率最高的密码子,则选择使用频率次高的密码子。
本发明还有一个目的在于,提供通过杆状病毒表达系统,高效表达密码子优化的基因。
本发明还有一个目的在于,提供一种猪圆环病毒基因工程疫苗。
本发明还有一个目的在于,提供一种猪圆环病毒基因工程苗的构建方法。
本发明在于获得GenBank中PCV2
ORF2表达Cap蛋白氨基酸一致序列的方法,对序列进行密码子优化的方法,并通过杆状病毒表达系统表达优化序列,经过高密度表达和灭活过程获得基因工程疫苗。
技术方案
本发明包括以下步骤:首先通过PCV2蛋白基因进行系统进化树分析,取其一致序列;对一致序列进行密码子优化;人工合成密码子优化序列YHsfORF2,并将其作为免疫原基因连接到HTA载体,构建重组质粒pFBHYHsfORF2;然后通过筛选获得阳性克隆并通过测序验证。之后将重组质粒转染Sf9细胞,获得重组杆状病毒vFBHYHsfORF2;通过经过高密度表达和灭活获得基因工程疫苗。
猪圆环病毒2型密码子优化的ORF2基因的重组病毒,其构建方法如下:
1) 人工合成猪圆环病毒2型密码子优化的ORF2基因,所述基因YHsfORF2序列为:SEQ.ID.NO.1;
2) 将所述基因YHsfORF2连入载体pMD-18T,获得pMD-18T- YHsfORF2;
3) 用SpeI/XholI双酶切将所述基因YHsfORF2克隆至载体pFastBacHTA中,获得重组质粒命名为pFBHYHsfORF2;
4) 将pFBHYHsfORF2质粒转化含helper质粒与Bacmid DNA的大肠埃希菌DH10Bac,获得含基因YHsfORF2的重组Bacmid DNA,转染Sf9细胞,获得重组杆状病毒vFBHYHsfORF2。
所述的重组病毒vFBHYHsfORF2可用于制备疫苗。
有益效果
本发明的特点和优点如下:
1、本发明应用的杆状病毒表达系统以其表达外源性蛋白产量高、抗原性好、产物易纯化等优点,在众多疫病的研究中得到广泛应用。
2、本发明应用的免疫原基因的密码子优化已成为提高疫苗免疫保护效果的有效策略之一。ORF2一致序列的优化,不仅提高了疫苗的交叉保护性,可以对免疫小鼠提供大于90%的抑毒率(图9),而且表达量提高了约3-4倍。
3、利用杆状病毒表达系统表达PCV2 ORF2优化基因,可形成病毒样颗粒。
4、本发明构建的重组病毒样颗粒,具有良好的的免疫原性和保护性。
5、本发明构建的重组病毒样颗粒疫苗不但安全性高,而且可以有效的预防PCV2感染及由PCV2感染而导致PCVAD的发生,是一种新型的安全有效的侯选疫苗。
四、附图说明
图1:重组质粒 pMD-18T- YHsfORF2 Spe I /XholI
双酶切鉴定图谱
图2:野毒株PCV2 ORF2的扩增:1:PCV2野毒; 2:阳性对照; 3:阴性对照
图3:重组质粒pFBHYHsfORF2和 pFBHsfORF2Spe I /XholI酶切鉴定图谱
1:pFBHORF2; 2:pFBHYHsfORF2; 3:M2000; 4:M15000
图4:正常的Sf9细胞与重组质粒转染后出现CPE的Sf9细胞:FBHYHsfORF2(a)、vFBHsfORF2(b)和细胞对照(c)
图5:重组杆状病毒vFBHYHsfORF2和vFBHsfORF2的间接免疫荧光图:vFBHYHsfORF2(a)、vFBHsfORF2(b)和细胞对照(c)
图6:vFBHYHsfORF2和vFBHsfORF2蛋白的免疫转印鉴定
1:Sf9细胞;
2:vHTA; 3:vFBHYHsfORF2; 4:vFBHORF2
图7:vFBHYHsfORF2和vFBHsfORF2病毒样粒子电镜图
图8:vFBHYHsfORF2和vFBHsfORF2在小鼠体内的ELISA抗体效价
图9:vFBHYHsfORF2和vFBHsfORF2在小鼠体内的抑毒率
五、具体实施方式
1
、表达外源基因的重组杆状病毒的构建
1)PCV2 ORF2基因的优化和合成
通过比对分析GenBank中PCV2的所有序列,取PCV2 ORF2表达氨基酸的一致序列,并通过密码子优化将PCV2 ORF2基因组改造为杆状病毒偏嗜密码子,并根据具体情况(GC含量、避免重复序列等)作适当调整,若不能使用频率最高的密码子,则选择使用频率次高的密码子。
优化的PCV2 ORF2基因定名为YHsfORF2基因,其序列(SEQ.ID.NO.1)为:
1 ATGACCTACCCAAGGAGACGTTACCGCAGGAGACGTCACAGGCCGAGATC
51
CCACCTCGGCCAAATCTTGAGAAGGAGACCATGGCTCGTGCACCCTCGTC
101
ACCGCTACAGGTGGCGTCGCAAGAACGGTATCTTCAACACAAGATTGTCA
151
CGTACATTCGGCTACACCATCAAGCGCACAACCGTTAAGACCCCATCGTG
201
GGCCGTGGACATGATGAGGTTCAACATCAACGATTTCCTGCCACCGGGTG
251
GCGGAAGCAACCCAAGATCAGTCCCGTTCGAGTACTACAGAATCCGTAAG
301
GTTAAGGTGGAATTCTGGCCCTGTTCCCCTATCACCCAGGGCGACAGGGG
351
AGTCGGTAGCTCAGCTGTTATCCTGGACGATAACTTCGTGACAAAGGCTA
401
CCGCCCTCACTTACGATCCCTACGTCAACTACTCGTCCAGACACACTATC
451 ACACAGCCTTTCTCCTACCACTCTCGTTACTTCACCCCCAAGCCTGTGCT
501
GGACTCAACTATCGATTACTTCCAACCTAACAACAAGCGCAACCAGTTGT
551
GGCTGAGGCTCCAAACAGCCGGTAACGTTGACCACGTGGGCCTCGGAACC
601
GCTTTCGAGAACTCTATCTACGATCAGGAATACAACATCCGCGTCACAAT
651
GTACGTTCAGTTCAGAGAGTTCAACCTCAAGGACCCACCCCTCAACCCCA
701 AG
根据HTA载体分析,在优化基因的两端分别加入酶切位点SpeI和XholI,人工合成含有酶切位点的优化基因并连入载体pMD-18T(Takara)(由GenScript公司完成),pMD-18T- YHsfORF2,测序验证正确。
蛋白序列(SEQ.ID.NO.2)如下:
1
MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLS
51
RTFGYTIKRTTVKTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRK
101 VKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTI
151
TQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGT
201
AFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPK
2)重组质粒pFBHYHsfORF2的构建与鉴定
利用双酶切位点(SpeI/XholI)将目的片段克隆至载体pFastBacHTA中;用SpeI/XholI双酶切(图2)及测序分析进行鉴定,将重组质粒命名为pFBHYHsfORF2。 如图2所示,电泳可见708bp的特异性片段,与预期结果一致,证明重组质粒构建成功。
3)重组质粒pFBHsfORF2的构建与鉴定
根据GenBank中收录的PCV2 Haian株和HBxz-PCV2a株的基因序列(GenBank accession number:FJ712216和FJ870968),利用Oligo6.0软件设计可以扩增PCV2 ORF2全基因的特异性引物,由上海英骏公司合成;以野毒PCV2 Haian株(参见郭容利, 何孔旺, 钟书霖等. 两株猪圆环病毒2型病毒的分离鉴定及其序列分析[J]. 江苏农业学报. 2009.25(5):1063-1067.)DNA作为模板,使用特异性引物扩增出PCV2 ORF2全基因片段并连入pMD-18T载体(购自Takara),酶切鉴定正确并送Invitrogen公司测序验证正确的质粒pMD-18T-WORF2;利用双酶切位点(SpeI/XholI)酶切pMD-18T-WORF2,将目的片段克隆至载体pFastBacHTA(购自Invitrogen,Bac-to-Bac® HT Vector Kit)中;用SpeI/XholI双酶切(图2)及测序分析进行鉴定,将重组质粒命名为pFBHsfORF2。由酶切结果和测序结果可知,重组质粒pFBHsfORF2构建成功。
4)重组Bacmid DNA的构建
将pFBHYHsfORF2和pFBHsfORF2质粒转化含helper质粒与Bacmid DNA的大肠埃希菌DH10Bac(购自invitrogen),使发生转座,产生分别含ORF2优化基因和野毒ORF2基因的重组Bacmid DNA,涂布于LB选择培养平板(卡那霉素50μg/ml,庆大霉素7μg/ml,四环素10μg/ml,IPTG20μg/ml,X-gal100μg/ml),37℃培养48h后挑选白色单克隆菌落,接种于加入卡那霉素、庆大霉素和四环素的LB培养基,37℃振摇18h之后提取小量Bacmid DNA;重组Bacmid DNA经PCR鉴定正确,冻存-20℃备用。
5)重组杆状病毒的获得
根据说明书使用Cellfection转染试剂(购自Invitrogen)将鉴定正确的pFBHYHsfORF2和pFBHsfORF2 Bacmid DNA转染Sf9细胞(ATCC),置27℃培养,每日观察细胞病变情况。pFBHYHsfORF2和pFBHsfORF2转染后72h,细胞出现明显的CPE,收获细胞上清,分装置无菌冻存管中,置4℃或-70℃保存,并将此病毒感染Sf9细胞进行扩增传代。传代感染细胞在感染后24-48h,观察到细胞变圆,直径增加,细胞核增大、膨胀,细胞折光性下降;72h细胞开始脱落、漂浮(图4)。
6)病毒样粒子的生物学特征研究
①间接免疫荧光
将重组病毒稀释后接种于长满单层的Sf9细胞,27℃培养,约48 h,弃去上清,用PBS洗涤一次,冰的无水乙醇4℃固定45 min,PBST洗涤3次;加入1:1000稀释的猪抗PCV2的阳性血清(购自VMRD),37℃作用1 h,用PBST洗涤3次;加入1:100 稀释的FITC标记的羊抗猪二抗(购自武汉博士德公司),37℃作用1 h,PBST洗涤3次;显微镜观察(图5)。结果显示,vFBHYHsfORF2和vFBHsfORF2感染的Sf9细胞的胞浆内均可以观察到特异性的绿色荧光,而正常Sf9细胞和接种HTA空载体表达产物的Sf9细胞均未观察到荧光,这也表明无论是野生型或经密码子优化的ORF2基因在Sf9细胞中均得到表达。
②Western blot分析
将重组病毒感染长满单层的Sf9 细胞, 同时设定正常Sf9细胞培养物为阴性对照。经过洗涤、裂解后进行常规SDS—PAGE 电泳,转印到硝纤膜后,10%脱脂乳封闭过夜;加入PCV2 ORF2的特异性单抗(参见曹彦琼、王先炜媛等.
猪圆环病毒2型Cap蛋白单克隆抗体的制备与鉴定[J]. 中国兽医科学. 2011.9(41):911-916.)(1 ∶1000 倍稀释) 室温作用2 h ; 洗涤3次,加入HRP 标记羊抗鼠IgG(购自武汉博士德)(1 ∶4000 倍稀释) ,室温作用1.5 h;充分洗涤后,加入化学发光法显色液(DAB)(购自武汉博士德), 观察特异性蛋白条带。结果表明,vFBHYHsfORF2和vFBHORF2出现与PCV2单克隆抗体特异性结合的条带,分子量约为28.7KD,而空白细胞和接种HTA空载体表达物的细胞均未出现条带,说明无论是野生型或经密码子优化的ORF2基因在Sf9细胞中均得到表达,且重组蛋白具有免疫学反应性(图6)。通过Bandscan和紫外风光光度计进行目的蛋白含量测定,结果表明vFBHYHsfORF2的表达量是vFBHsfORF2的3-4倍。
③病毒样粒子的粗纯化与电镜观察
收集病变细胞,用PBS洗两次,1000 r/min 离心15 min,沉淀重悬于50 mmol/l NaHCO3中,超声波裂解后,24 000 r/min离心15 min,上清液中含有病毒样颗粒。收集的上清中加入PEG-6000至终浓度10%,4℃ 搅拌过夜进行沉淀,然后 10000g/min离心20 min ,沉淀重悬于PBS中即为病毒样颗粒的浓缩粗提物。加入1:100稀释的猪抗PCV2 ORF2的特异性单克隆抗体(参见曹彦琼、王先炜媛等. 猪圆环病毒2型Cap蛋白单克隆抗体的制备与鉴定[J]. 中国兽医科学. 2011.9(41):911-916.)37℃ 作用1 h 后再4℃过夜,以12 000 r/min 离心90 min,收集沉淀即为纯化的病毒样颗粒。取沉淀重悬于少量ddH2O中,用3%的磷钨酸负染后经透射电镜观察。如图7所示,vFBHYHsfORF2和vFBHORF2均可见17nm左右的病毒样颗粒,这也进一步表明密码子优化没有影响其生物学特性。
5)小鼠免疫保护实验
4周龄的BALB/C小鼠20只,随机分为3组,每组5只。第一组免疫灭活的vFBHYHsfORF2,第2组免疫灭活的vFBHsfORF2,第3组注射细胞培养基作为对照组,各组均加入等体积弗氏不完全佐剂乳化,腹腔注射200μL/只,免疫前和免疫后1、2和3周尾静脉采血,分离血清用原核表达纯化的PCV2 Cap蛋白包被酶标板检测BALB/C小鼠血清中的PCV2抗体水平。具体ELISA方法如下:由ELISA方阵试验可以得出,当抗原包被浓度为2.5 μg/mL,抗体稀释100倍,一抗37℃分别孵育1.0 h, 酶标二抗37℃孵育45 min,加TMB-H2O2(四甲基联苯胺-过氧化氢)溶液显色,37℃反应10-15 min。加2M H2SO4溶液,终止反应,50 μL/孔。反应条件比较合适,此时P/N值较大,且阴性血清的OD值较小,即非特异性比较小。结果判定:P - N >0.15 时实验有效。(样品OD值- N )/( P - N )≥0.45阳性;(样品OD值- N )/ (P - N )< 0.45阴性。其中P 表示:阳性对照平均值; N 表示:阴性对照平均值。结果可见,vFBHYHsfORF2和vFBHsfORF2免疫后7天,几乎所有小鼠抗体转阳,免疫后1-3周抗体水平不断升高,而免疫细胞培养基组不产生抗体,这也说明vFBHYHsfORF2和vFBHsfORF2可以刺激小鼠产生特异性抗体,且vFBHYHsfORF2诱导产生的抗体值显著高于vFBHsfORF2,免疫效果较好
所有上述3组于免疫后第21天每只腹腔注射0.2ml的PCV2 2010AHCY(105TCID50)(参见王小敏,张文文,何孔旺等..2009-2010年华东地区猪圆环病毒2型的基因型分析[J]. 江苏农业学报. 2011.27(5):1037-1042.)进行攻毒,攻毒后1、2、3和4周尾静脉采血,利用Real-time PCR方法检测血清样品中的PCV2病毒拷贝数,根据公式【(攻毒对照组病毒平均拷贝数-免疫组病毒平均拷贝数)/攻毒对照组病毒平均拷贝数】X100%,计算灭活的重组病毒vFBHYHsfORF2和vFBHYHORF2所制疫苗对病毒复制产生的抑制率。如图9所示,定量检测结果表明,免疫vFBHYHsfORF2和vFBHYHORF2的小鼠攻毒后血清样品中的PCV2病毒拷贝数与只免疫细胞培养基的攻毒对照组经t检验差异显著,重组病毒vFBHYHsfORF2对PCV2病毒复制的抑制率最佳,达到90%以上,优于vFBHYHORF2组。
本发明成功构建了表达密码子优化的PCV2 Cap蛋白的重组杆状病毒,通过免疫荧光和Western-blot试验验证了其高效表达,且表达的Cap蛋白具有生物学活性,可以与PCV2的阳性血清发生特异性反应。电镜观察进一步证明重组杆状病毒表达的密码子优化的Cap蛋白和未优化的野毒Cap蛋白类似,都可以自我组装形成17nm左右的病毒样颗粒,说明密码子优化并未改变其生物学活性。小鼠免疫保护试验结果表明,重组病毒vFBHYHsfORF2和vFBHYHORF2对小鼠体内的PCV2复制具有很好的抑制作用,且vFBHYHsfORF2对PCV2病毒的抑制效果要优于vFBHYHORF2。这也表明本发明构建的重组疫苗不但安全性高而且可以有效的预防PCV2感染,是一种新型的安全有效的侯选疫苗。
SEQUENCE LISTING
<110> 江苏省农业科学院
<120> 猪圆环病毒2型密码子优化的ORF2基因的重组病毒样粒子
<130> 0
<160> 2
<170> PatentIn
version 3.1
<210> 1
<211> 702
<212> DNA
<213> 人工
<220>
<221> YHsfORF2基因
<222> (1)..(702)
<223>
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aagaacggta tcttcaacac
aagattgtca cgtacattcg gctacaccat caagcgcaca 180
accgttaaga ccccatcgtg
ggccgtggac atgatgaggt tcaacatcaa cgatttcctg 240
ccaccgggtg gcggaagcaa
cccaagatca gtcccgttcg agtactacag aatccgtaag 300
gttaaggtgg aattctggcc
ctgttcccct atcacccagg gcgacagggg agtcggtagc 360
tcagctgtta tcctggacga
taacttcgtg acaaaggcta ccgccctcac ttacgatccc 420
tacgtcaact actcgtccag
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ttcaccccca agcctgtgct
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702
<210> 2
<211> 234
<212> PRT
<213> 人工
<220>
<221> YHsfORF2基因蛋白氨基酸序列
<222> (1)..(234)
<223>
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Claims (1)
1.猪圆环病毒2型密码子优化的ORF2基因的重组病毒,其构建方法如下:
1)人工合成猪圆环病毒2型密码子优化的ORF2基因,命名为YHsfORF2基因,所述基因YHsfORF2序列为:SEQ.ID.NO.1;
2)将所述基因YHsfORF2连入载体pMD-18T,获得pMD-18T-YHsfORF2;
3)用SpeI/XholI双酶切将所述基因YHsfORF2克隆至载体pFastBacHTA中,获得重组质粒命名为pFBHYHsfORF2;
4)将pFBHYHsfORF2质粒转化含helper质粒与Bacmid的大肠埃希菌DH10Bac,获得含基因YHsfORF2的重组Bacmid DNA,转染Sf9细胞,获得重组杆状病毒vFBHYHsfORF2。
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CN104498439B (zh) * | 2014-11-28 | 2017-10-03 | 湖南农业大学 | 杂交瘤细胞、pcv2单克隆抗体及其应用 |
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CN105999255A (zh) * | 2016-05-19 | 2016-10-12 | 华南农业大学 | 一种猪圆环病毒ii型病毒样颗粒疫苗及其制备方法和应用 |
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