CN1562365A - SARS vaccine of adenovirus carrier and preparation method, application of coronavirus S gene - Google Patents

SARS vaccine of adenovirus carrier and preparation method, application of coronavirus S gene Download PDF

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CN1562365A
CN1562365A CN 200410044285 CN200410044285A CN1562365A CN 1562365 A CN1562365 A CN 1562365A CN 200410044285 CN200410044285 CN 200410044285 CN 200410044285 A CN200410044285 A CN 200410044285A CN 1562365 A CN1562365 A CN 1562365A
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sars
gene
adenovirus
vaccine
coronavirus
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CN1276777C (en
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黄文林
曾益新
汪健
刘然义
黄嘉凌
黄必军
赖坤
吴立志
梁志慧
柯妙拉
吴秀菊
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中山大学肿瘤防治中心
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Abstract

本发明属于生物基因工程领域,具体涉及腺病毒载体SARS疫苗,其制备方法以及相关冠状病毒S基因在制备预防非典型肺炎(SARS)疫苗方面的应用。 The present invention belongs to the field of genetic engineering, particularly relates to adenoviral vector SARS vaccines, their preparation and coronavirus S genes in SARS (SARS) on vaccines for the prophylaxis. 通过生物工程手段,将相关冠状病毒S基因与缺陷型腺病毒重组结合,使保护性免疫原蛋白或多肽在其中表达,经过扩增培养、纯化、制剂而制成一种能引起粘膜免疫原性的基因疫苗,诱导呼吸道粘膜免疫反应,使机体产生相应抗体,防止病毒侵染。 By means of biological engineering, the coronavirus S gene in combination with deficient recombinant adenovirus, the protective immunogen protein or polypeptide expressed therein, through expansion culture, purification, and formulation to prepare a mucosal immunogenicity can cause the gene vaccine, respiratory mucosal immune response induced by the body to produce antibodies against the virus infection. 本发明与传统的灭活病毒颗粒疫苗比较,安全性高,使用方便,不受肌肉注射等特定条件限制,具有广泛的临床应用前景。 Specific conditions of the present invention, compared with conventional inactivated virus particle vaccine, safe, easy to use, without limitation intramuscular, have broad clinical applications.

Description

腺病毒载体SARS疫苗及其制备方法, 冠状病毒S基因的应用 Adenoviral vector vaccine and its preparation method SARS coronavirus S gene with

一、技术领域本发明属于生物基因工程领域,具体涉及腺病毒载体SARS疫苗,其制备方法以及揭示相关冠状病毒S基因在制备预防非典型肺炎(SARS)疫苗方面的应用。 I. Technical Field The present invention belongs to the field of genetic engineering, particularly relates to adenoviral vector SARS vaccines, their preparation and coronavirus S gene disclosed in the application of the preparation of vaccines against SARS (SARS).

二、背景技术 Second, the technical background

中国广东地区从去年末开始爆发非典型肺炎(atypical pneumonia),发病急,传染性强,抗生素治疗无效。 China Guangdong region from the end of last year, the outbreak of atypical pneumonia (atypical pneumonia), the incidence of acute, contagious, antibiotic treatment ineffective. 目前,此病已传播至30多个国家和地区,WHO已将之命名为严重急性的呼吸道综合症(severe acuterespiratory syndrome,SARS). At present, the disease has spread to more than 30 countries and regions, WHO has been named for severe acute respiratory syndrome (severe acuterespiratory syndrome, SARS).

目前,世界许多国家的科学家均从不同病人血清中独立分离到新型的冠状病毒(coronavirus),病毒基因序列分析结果表明,它们与已知的冠状病毒有50%~60%的同源性,世界卫生组织(WHO)已经公布,引起SARS的病原体是冠状病毒的变异株。 At present, scientists in many countries around the world independently isolated from different patient serum to a new type of coronavirus (coronavirus), viral gene sequence analysis showed that they are known coronavirus 50% to 60% homology, world Health Organization (WHO) has announced that the SARS pathogen is causing mutant coronavirus.

冠状病毒是一种非节段性正链的RNA病毒,其基因组近30kb,可以在人和动物中传播,主要感染人和动物的呼吸系统,冠状病毒颗粒是一种具有内核心及囊膜包被地病毒,共有四种结构蛋白:刺突蛋白(spike,S),膜蛋白(membrance,M),囊膜蛋白(envelop,E),及核蛋白(nucleoprotein,N)。 Is a non-segmental coronavirus positive strand RNA viruses whose genome near 30KB, can be spread in humans and animals, primary infection of the respiratory system in humans and animals, a coronavirus having an inner core particles and coating the capsule virus, there are four structural proteins: a spike protein (spike, S), membrane protein (membrance, M), envelope proteins (envelop, E), and nucleoprotein (nucleoprotein, N). 由于冠状病毒是一种RNA病毒,十分不稳定,容易发生突变以逃避宿主的免疫监督与排斥。 Because coronavirus is an RNA virus, very unstable and prone to mutation to evade the host's immune surveillance and exclusion. 因此,必须寻找到SARS相关冠状病毒中稳定性好且具有免疫保护作用的抗原,以进行相关疫苗的研制。 Therefore, we must find a good SARS-associated coronavirus in stability and has a protective immune antigen for the development of relevant vaccines.

目前较少应用灭活的病毒颗粒疫苗,因为全病毒颗粒携带了病毒全基因组,安全性顾虑较大。 Currently rarely used viral particles inactivated vaccine, because the whole virus particles carrying the virus genome, larger security concerns. 虽然既往的冠状病毒易在体外培养获得,但此次SARS相关的冠状病毒毒性极大且遗传背景不完全清楚,因此大规模制备冠状病毒颗粒不可行。 Although previous coronavirus easy to obtain in vitro, but the SARS-associated coronavirus highly toxic and genetic background is not entirely clear, and therefore large scale production of coronavirus particles is not feasible. 基因工程技术的发展为亚单位疫苗的开发提供了极大的便利,而且亚单位疫苗的可操作性及生物安全性好。 The development of genetic engineering technology provides great convenience for the development of subunit vaccines, biological and operability and safety of subunit vaccines is good. 若能筛选到具有免疫原性的病毒抗原决定簇,结合基因工程技术,可方便地对抗原决定簇进行改造,以增强其稳定性、免疫原性、生物安全性。 If viral antigen screening immunogenic determinants, combined with genetic engineering techniques, can easily be modified to epitopes in order to enhance its stability, immunogenicity, biological safety. 显然,借助基因工程技术,可以非常方便地制备有效的亚单位疫苗。 Clearly, the aid of genetic engineering techniques, can easily effective subunit vaccine preparation.

根据目前研究显示,冠状病毒已知的四种结构蛋白中,刺突蛋白(spike S)是具有诱导保护性免疫反应的结构蛋白,部分学者的研究结构已经证实了冠状病毒刺突蛋白C末端为其抗原决定族所在,同时,SARS相关冠状病毒通过呼吸道引起感染,而目前仍未有可以预防SARS的疫苗。 The present study shows that the four known coronavirus structural proteins, spike protein (spike S) is a structural protein of inducing a protective immune response, some scholars studies have confirmed the structure of coronavirus spike protein C-terminus antigenic determinants where, at the same time, SARS-associated coronavirus infection caused by respiratory tract, but has yet to have a vaccine to prevent SARS.

另外,资料显示,腺病毒本身很容易感染呼吸道粘膜上皮,较易诱导呼吸道粘膜免疫反应,并且腺病毒作载体表达的免疫原在宿主细胞中表达、加工、折叠、修饰、提呈,基本上保持了免疫原的天然构象,生物活性高。 Further, the data show, the adenovirus itself is susceptible to respiratory mucosa, respiratory mucosa easier to induce an immune response, and immune adenoviral expression vector for expression in a host cell original, processing, folding, modification, presenting substantially maintained immunogen native conformation, high biological activity.

所述条件为利用缺陷型腺病毒作为载体研制和生产腺病毒SARS疫苗提供了坚实的理论基础。 The condition provides a solid theoretical foundation for the use of defective adenovirus as a vector development and production of adenovirus SARS vaccine.

三、发明内容 III. SUMMARY OF THE INVENTION

本发明的第一个目的在于提供一种能预防流行性疾病“非典型肺炎”的腺病毒SARS疫苗,更好的防止“非典型肺炎”的发生和传播;本发明另外的目的在于提供一种利用缺陷型腺病毒作为载体,通过基因克隆、重组等手段,制备腺病毒载体SARS疫苗的方法以及揭示SARS相关冠状病毒S基因在制备疫苗中的应用。 A first object of the present invention is to provide adenoviral vaccine can prevent SARS epidemic "SARS", the better to prevent "SARS" the occurrence and spread; a further object of the present invention to provide a using as deficient adenoviral vector, by cloning, recombinant and other means, adenoviral vector disclosed SARS vaccine preparation and use of vaccines SARS-associated coronavirus S gene in the manufacture.

本发明的目的是这样实现的:通过生物工程手段,将SARS相关冠状病毒S基因(冠状病毒共有四个结构基因,S基因为其中之一,见下文详述)与缺陷型腺病毒重组结合,构成一种能引起粘膜免疫原性的基因疫苗。 Object of the present invention is achieved: by means of biological engineering, the S gene of SARS-associated coronavirus (CoV total of four structural gene, wherein one of the S gene, see detailed description below) in combination with deficient recombinant adenovirus, constitute a gene vaccine can cause mucosal immunogenicity.

所用Spike基因片段序列为:使用Gene bank中所公布的S基因序列(Gene bank查询号:gbAY278554.2)作为模版,根据序列设计PCR引物如下:v1 GGTCTAGAGT TGTGGTTTCA AGTGATv2 TTTCTAGACC ATGGGTTGTG TCCTTGCTV3 TTTCTAGACC ATGGCATATA GGTTCAATGV4 TAGGTACCAA TGCCAGTAGT GGTGV5 TTGGTACCTC CGCCTCGACT TTV6 CCGGTACCAT AAGTTCGTTT ATGTGT其中V1和V4为一对,扩增S基因N端片段;V2和V5为一对引物,扩增S基因M区片段;V3和V6为一对引物,扩增S基因C端片段(如图1),扩增后的结构见图2。 As used Spike gene fragment sequence: Using the S gene sequence (Gene bank accession number: gbAY278554.2) Gene bank as published as a template, in accordance with the sequence PCR primers were designed as follows: v1 GGTCTAGAGT TGTGGTTTCA AGTGATv2 TTTCTAGACC ATGGGTTGTG TCCTTGCTV3 TTTCTAGACC ATGGCATATA GGTTCAATGV4 TAGGTACCAA TGCCAGTAGT GGTGV5 TTGGTACCTC CGCCTCGACT TTV6 CCGGTACCAT AAGTTCGTTT ATGTGT wherein a pair of V1 and V4, the S gene amplified N-terminal fragment; V2 and V5 as a pair of primers, M S gene amplified fragment; V3 and V6 to a pair of primers, amplification of gene S C-terminal fragment (FIG. 1), the structure of Figure 2 after amplification.

腺病毒SARS疫苗的制备:首先,收集合分离康复病人血清,通过分离提取得到冠状病毒总RNA,通过反转录、测序、挑选等步骤得到S基因。 Preparation of adenovirus SARS vaccine: First, collect co rehabilitation patient serum separation, total RNA was obtained by separation and extraction of coronavirus, the S gene obtained by reverse transcription, sequencing, selection and other steps. 接着,将S基因克隆到pShuttle质粒得到克隆体(保藏单位:中国典型培养物保藏中心;保藏日期:2003年5月17日;保藏号:CCTCC M 203036 E.coliDH5a/pShuttle-SN以及CCTCC M 203037 E.coli DH5a/pShuttle-SC),再将其与腺病毒骨架质粒(pAdeno-XTM)连接(其中pShuttle、pAdeno-X均购自CLONTECH Laboratory,Inc,USA)。 Subsequently, the S gene was cloned into the plasmid pShuttle resulting clone (Accession: China Center for Type Culture Collection; Date of Deposit: May 17, 2003; deposit number: CCTCC M 203036 E.coliDH5a / pShuttle-SN and CCTCC M 203037 E.coli DH5a / pShuttle-SC), which is then connected to the adenoviral backbone plasmid (pAdeno-XTM) (wherein pShuttle, pAdeno-X were purchased from CLONTECH Laboratory, Inc, USA). 连接后共同转染293细胞,经进一步的纯化鉴定后,用293细胞大量扩增,收集病毒液,分离纯化,即为腺病毒SARS疫苗。 After co-transfected 293 cells is connected, after further purification identification, a large number of amplification in 293 cells, the virus was collected, separated and purified, namely adenovirus SARS vaccine. 它可以制成喷雾剂或其它形式的药剂。 It may be made of other forms of agents or spray. 主要的技术路线见图4。 The main technical route shown in Figure 4.

本发明是一种基因工程疫苗,即是使用缺陷型腺病毒作为载体的基因疫苗,它利用本身很容易感染呼吸道粘膜上皮的腺病毒作为载体,使保护性免疫原蛋白或多肽在其中表达,诱导呼吸道粘膜免疫反应;通过诱导粘膜免疫性反应,使机体产生相应抗体,防止病毒侵染。 The present invention is a genetically engineered vaccine, i.e., using a gene-defective adenovirus vaccine vector, which per se is susceptible to respiratory mucosal epithelium as adenoviral vectors, the protective immunogen protein or polypeptide expressed therein, induced respiratory mucosal immune response; mucosal immunity by inducing a reaction, the body to produce antibodies against the virus infection. 本发明与传统的灭活病毒颗粒疫苗比较,它安全性高,并且使用方便,不受肌肉注射等特定条件限制。 Present invention compared with the conventional vaccine inactivated virus particles, it is safe, and easy to use, certain other conditions are not restricted by intramuscular injection.

目前,SARS正在世界各地快速传播,作为一种病毒性传染病,眼下尚未找到可以有效治疗的药物,此种情况下,预防是最好的手段。 Currently, SARS spread rapidly around the world, as a viral infection, has not yet found the drug can now be effectively treated, in this case, prevention is the best means. 现已证实SARS相关冠状病毒刺突蛋白C末端为其抗原决定族所在。 It has been proved antigenic determinants where the SARS-associated coronavirus spike protein for its C-terminus. 因此,本发明正是根据此发现,合成SARS相关冠状病毒刺突蛋白基因,将其克隆到腺病毒载体,经过扩增培养、纯化、制剂而成,能有效诱导粘膜产生抗体,产生体液免疫防止病毒侵染机体,具有广泛的临床应用前景。 Accordingly, the present invention is based on this discovery, the synthesis of SARS-associated coronavirus spike protein gene was cloned into an adenovirus vector, culturing after amplification, purification, formulation is made effective to produce an antibody capable of inducing mucosal, humoral immunity to prevent virus infection of the body, with a wide range of clinical applications.

四、附图说明 IV BRIEF DESCRIPTION

图1是S基因片段扩增示意图。 Figure 1 is a schematic view of the S gene amplified fragment.

图2是带有S基因(Spike-S)的重组缺陷型腺病毒结构图。 FIG 2 is a recombinant defective adenovirus having a configuration diagram of the S gene (Spike-S) of.

图3是pShuttle-SC、pShuttle-SM、pShuttle-SN重组体酶切结果。 FIG 3 is pShuttle-SC, pShuttle-SM, pShuttle-SN recombinant enzyme results.

图4是pShuttle-SC、pShuttle-SM、pShuttle-SN重组体测序结果。 FIG 4 is a pShuttle-SC, pShuttle-SM, pShuttle-SN recombinant sequencing results.

图5是RT-PCR检测Ad-SN和Ad-SM的表达试验结果图6是Western blot检测Ad-SN的表达试验结果图7是Ad-SN注射或呼吸道免疫均诱导雌性大鼠产生特异性抗体(IgG)的体内表达试验结果图8是Ad-SN注射或呼吸道免疫均诱导雄性大鼠产生特异性抗体(IgG)的体内表达试验结果图9是本发明疫苗制备的技术路线图。 FIG 5 is a test result of FIG expression by RT-PCR, and Ad-SN Ad-SM 6 is a test result of the expression of Ad-SN FIG. 7 is a Western blot analysis Ad-SN are injected or induced respiratory female rats immunized to produce antibodies (IgG) the results of the in vivo expression of FIG. 8 is expressed in vivo test results of FIG Ad-SN are injected or respiratory immunity was induced in male rats to produce specific antibody (IgG) 9 is a roadmap of the vaccine preparation of the present invention.

五、具体实施方式 V. DETAILED DESCRIPTION

以下将通过具体实施例来进一步说明本发明。 The following will further illustrate specific embodiments of the present invention.

实施例1腺病毒载体SARS疫苗的制备方法:腺病毒载体SARS疫苗的制备分为前期构建和后期扩增二部分:前期构建:取得SARS相关冠状病毒spike(SF、SN、SM、SC)基因后,用PCR方法进行扩增,经PCR后,用Xbal+Kpnl 37℃酶切,同时用此酶酶切pShuttle,连接,转化大肠杆菌DH5a,利用卡那霉素(KanR)抗性筛选阳性克隆,培养、纯化后得到pSF/SN/SM/SC-Shuttle,用1-Ceul+P1-Scel酶切,同时用此酶酶切PAdeno-XTM,酶切后连接,转化大肠杆菌DH5a,利用氨苄(Amp+)抗性筛选阳性克隆即得pAd-SF/SN/SM/SC。 Example 1 Preparation method of adenoviral vector vaccine embodiment of SARS: after obtaining the SARS-associated coronavirus spike (SF, SN, SM, SC) Gene: Construction of pre-: Preparation of adenovirus vector construct SARS vaccine pre and post-amplification is divided into two parts , by PCR amplification, by PCR after digestion with Xbal + Kpnl 37 ℃, while the pShuttle digested with this enzyme, ligation, transformation of E. coli DH5a, kanamycin (the KanR) screening positive clones resistant, culture, after purification pSF / SN / SM / SC-Shuttle, with 1-Ceul + P1-Scel enzyme, while the enzyme digested with pAdeno-XTM, the ligated, transformed into E. coli DH5a, using ampicillin (Amp + ) to obtain positive clones resistant pAd-SF / SN / SM / SC.

扩大培养:得到pAd-SF/SN/SM/SC后,再经Pacl酶切、质粒经线化后转染包装细胞,包装细胞为整合了C亚类5型腺病毒(Ad5)E1区基因的细胞系(株),例如293细胞。 Expanding culture: obtained pAd-SF / SN / SM / SC after then by Pacl digested plasmid was transfected into a packaging cell warps, the packaging cell is a C alkylene integrated type 5 adenovirus (the Ad5) gene region E1 cells line (line), for example, 293 cells. 经过空斑筛选,PCR鉴定为Ad-SF/SN/SM/SC后,大量培养293细胞,用Ad-SF/SN/SM/SC感染293细胞,经氯化铯分离纯化,制剂等步骤,即得到腺病毒SARS疫苗。 After plaque screening, PCR identification of Ad-SF / SN / SM / SC, a large number of cultured 293 cells with Ad-SF / SN / SM / SC 293 infected cells, was purified by cesium chloride, preparations step, i.e., adenovirus get SARS vaccine.

SARS疫苗包含SARS相关冠状病毒S基因和和缺陷型腺病毒。 SARS vaccine containing SARS-associated coronavirus gene and S and deficient adenovirus. 缺陷型腺病毒为E1区完全缺失的C亚类的5型腺病毒,即Ad5。 Deficient adenovirus E1 region is completely deleted subclass C adenovirus type 5, i.e., Ad5. 该缺陷型腺病毒的E3区可以为完全缺失或部分缺失或不缺失。 The deficient adenoviral E3 region may be partially or completely deleted or without deletion. 缺陷型腺病毒内装CMV启动子和BGHpolyA。 Built-deficient adenovirus The CMV promoter and BGHpolyA.

Ad-SF/SN/SM/SC克隆到腺病毒载体中的为SARS相关冠状病毒S基因全长。 Ad-SF / SN / SM / SC is cloned into a SARS-associated coronavirus S gene of the full-length adenoviral vector.

克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S1结构域在内的序列。 Cloned into the adenoviral vector comprises a sequence including the S1 domain of SARS-associated coronavirus S gene.

克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S2结构域在内的序列。 Cloned into the adenoviral vector comprises a sequence including the S2 domain of SARS-associated coronavirus S gene.

克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S1、S2结构域在内的序列(碱基号为:49~3585)。 Cloned into the adenoviral vector is a SARS-associated coronavirus S gene comprising S1, S2 domain including the sequence (number of bases: 49 to 3585).

克隆到腺病毒载体中的为SARS相关冠状病毒S基因跨膜区(碱基号为:3686~3648)及C端片段。 Cloned into the adenoviral vector is a SARS-associated coronavirus S gene transmembrane region (base numbers: 3686 ~ 3648) and a C-terminal fragment.

实施例2克隆到腺病毒载体中的为SARS相关冠状病毒S基因N端片段。 Example 2 Cloning of the SARS-associated coronavirus S gene N terminal fragment of the adenoviral vector. 其余同实施例1。 Remaining the same as in Example 1.

实施例3克隆到腺病毒载体中的为SARS相关冠状病毒S基因中间片段。 Example 3 Cloning of the S gene of coronavirus is SARS-associated middle segment of the adenoviral vector. 其余同实施例1。 Remaining the same as in Example 1.

实施例4克隆到腺病毒载体中的为SARS相关冠状病毒S基因C端片段。 Cloned into the adenoviral vector is a SARS-associated coronavirus S gene C-terminal fragment of the fourth embodiment.

其余同实施例1。 Remaining the same as in Example 1.

实施例5S基因转化pShuttle质粒和鉴定:用Xbal+Kpnl 37℃水浴条件酶切,同时用此酶酶切pShuttle,连接,转化大肠杆菌DH5a,培养,利用卡那霉素(KanR)抗性筛选阳性克隆,分别得到pShuttle-SC、pShuttle-SM、pShuttle-SN,通过常规琼脂凝胶电泳鉴定和检测序列鉴定,结果见图3、图4。 Example 5S gene transfer and identification of plasmid pShuttle: 37 [deg.] C with a water bath Kpnl + Xbal digestion conditions, while pShuttle digested with this enzyme, ligation, transformation of E. coli DH5a, culture, kanamycin (the KanR) positive resistance screening clones, respectively pShuttle-SC, pShuttle-SM, pShuttle-SN, via conventional agar gel electrophoresis and detection sequence identification, results shown in Figure 3, Figure 4.

实施例6对克隆得到的Ad-SN/SM进行体外表达试验。 Example 6 clones obtained Ad-SN / SM in vitro expression experiments.

用2.5~40 MOI感染VeroE6细胞,弃去病毒悬液,加入细胞培养液,37℃、5%CO2条件下培养,于感染后48h收集培养细胞上清,分别用RT-PCR法检测Ad-Sn和Ad-Snm的表达水平,用Western blot法检测Ad-Sn的表达水平,结果见图5、图6。 With 2.5 ~ 40 MOI VeroE6 infected cells, the virus suspension was discarded, the cell culture medium was added, cultured at 37 ℃, 5% CO2 conditions, 48h in the culture supernatants collected after in infection with Ad-Sn were detected by RT-PCR and Ad-Snm expression level detected in Ad-Sn expression levels by Western blot results shown in Figure 5, FIG. 6.

实施例7对克隆得到的Ad-SN进行体内表达试验。 Example 7 Ad-SN clones was subjected to in vivo expression experiments.

将试验对象按照下述方式分组:Ad-Sn滴鼻组、Ad-lacZ滴鼻组(对照,先用3%戊巴比妥腹注麻醉,滴鼻0.5mL/只)、Ad-Sn尾静脉注射组、Ad-lacZ尾静脉注射组(对照,尾静脉注射给药0.5mL/只)、空白对照组(不作任何处理)。 The test objects grouped in the following manner: Ad-Sn intranasal group, Ad-lacZ intranasal group (control, first with 3% pentobarbital anesthesia abdominal injection, intranasal 0.5mL / only), Ad-Sn tail vein injected group, Ad-lacZ injected intravenously (control, tail vein injection 0.5mL / only) blank control group (no treatment). 然后按照下述方式给药:每周给药一次,连续三周;从第一次给药后一周开始取血,最后一次给药2周后处死动物取血和组织,用ELISA法测定大鼠抗SARS IgG浓度,结果见图7、图8。 Then the following manner of administration: administered once a week for three weeks; bled one week after the start from the first administration, 2 weeks after the last administration the animals were sacrificed and blood tissues of rats was measured by ELISA anti-SARS IgG concentration, the results shown in Figure 7, FIG.

实施例8绿猴肾细胞(Vero E6)细胞免受SARS攻击1.接种Vero E6细胞到96孔板,2×104/孔。 Example 8 green monkey kidney cells (Vero E6) SARS attack cells from 1 to Vero E6 cells were seeded a 96 well plate, 2 × 104 / hole embodiment.

2. 24小时后,接种腺病毒SARS疫苗,方法:用1640培养液按4倍稀释病毒原液,接种时先将96孔板中培养液吸出弃去,PBS清洗孔一遍后,加入不同稀释度的病毒液,每一个稀释度加5个孔,50μL/孔。 2. After 24 hours, the adenovirus SARS vaccine inoculation, method: press 4 with 1640-fold diluted viral stock solution, when a 96 well plate were seeded first aspirated broth was discarded, the wells were washed once with PBS, were added different dilutions virus solution, each dilution plus five holes, 50μL / hole. 同时以1640培养液(不含病毒)为阴性对照。 While 1640 (virus-free) as a negative control. 37℃,5%CO2,饱和湿度条件下孵育1小时后,加入含5%新生牛血清的1640培养液,200μL/孔,37℃,5%CO2,饱和湿度条件下培养。 After 1 hour incubation at 37 ℃, 5% CO2, saturated humidity, was added 1640 containing 5% newborn calf serum, 200μL / well and cultured at 37 ℃, 5% CO2, saturated humidity.

3. 24小时后,接种SARS病毒,方法:1640培养液稀释SARS病毒至100TCID50,接种时先将96孔板中培养液吸出弃去,PBS清洗孔一遍后,加入不同稀释度的病毒液,每一个稀释度加5个孔,50μL/孔。 3. After 24 hours, the SARS virus inoculation, method: 1640 dilution to the SARS virus 100TCID50, when the first seeded in 96-well plates were aspirated and the culture was discarded, the wells were washed once with PBS, were added different dilutions of the virus solution, each a dilution plus five holes, 50μL / hole. 同时以1640培养液(不含病毒)为阴性对照。 While 1640 (virus-free) as a negative control. 37℃,5%CO2,饱和湿度条件下孵育1小时后,加入含5%新生牛血清的1640培养液,200μL/孔,37℃,5%CO2,饱和湿度条件下培养。 After 1 hour incubation at 37 ℃, 5% CO2, saturated humidity, was added 1640 containing 5% newborn calf serum, 200μL / well and cultured at 37 ℃, 5% CO2, saturated humidity.

4.此后每隔12到24小时观察并记录细胞病变情况,计算结果时各稀释度的每孔细胞病变评分相加,得出细胞病变指数,取各组平均数,如下: 4. After every 12 to 24 hours the cells were observed and recorded lesions, the calculation result of each dilution are added per well cytopathic ratings, stars cytopathic index, the average of each group, as follows:

<110>中山大学肿瘤防治中心<120>腺病毒载体SARS疫苗及其制备方法,冠状病毒S基因的应用<160>4<210>1<211>3780<212>DNA<213>病毒种<220> & Lt; 110 & gt; Cancer Center of Zhongshan University & lt; 120 & gt; adenoviral vector SARS vaccine and its preparation method, coronavirus S gene with & lt; 160 & gt; 4 & lt; 210 & gt; 1 & lt; 211 & gt; 3780 & lt; 212 & gt; DNA & lt; 213 & gt; virus species & lt; 220 & gt;

<221>CDS<222>(1)…(3780)<400>1atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600 tgtccgtggt 300tgggtttttg 1atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt; & lt; 221 & gt; CDS & lt; 222 & gt; (1) ... (3780) & lt; 400 & gt gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600

caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat1320aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc1380tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat1440 caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat1320aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc1380tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat1440 tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtttctga tttcactgat 1680tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgcgc ttttgggggt 1740gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800gttaactgca ctgatgtttc tacagcaatt catgcagatc aactcacacc agcttggcgc 1860atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920 tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtttctga tttcactgat 1680tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgcgc ttttgggggt 1740gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800gttaactgca ctgatgtttc tacagcaatt catgcagatc aactcacacc agcttggcgc 1860atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920

catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac1980catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct2040ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca2280cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc2460gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatggactt2520acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640gctatgcaaatggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag2700aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760acaacaacat caactgcat catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac1980catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct2040ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca2280cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc2460gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatggactt2520acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640gctatgcaaatggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag2700aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760acaacaacat caactgcat t gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880 t gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880 ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240 ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240

ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360gatcctctgc aacctgagct tgactcattc aaagaagagc tggacaagta cttcaaaaat 3420catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660agttgttgca gttgcctcaa gggtgcatgctcttgtggtt cttgctgcaa gtttgatgag3720 ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300aatacatttg 3660agttgttgca gttgcctcaa gggtgcatgctcttgtggtt tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360gatcctctgc aacctgagct tgactcattc aaagaagagc tggacaagta cttcaaaaat 3420catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact cttgctgcaa gtttgatgag3720 <210>2<211>50<212>DNA<213>人工序列<220> & Lt; 210 & gt; 2 & lt; 211 & gt; 50 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>根据spike基因碱基序列设计的用于PCR扩增spike基因N端的引物(一对)<400>2ggtctagagt tgtggtttca agtgattaggtaccaa tgccagtagt ggtg<210>3 & Lt; 223 & gt; the N-terminus of the spike gene amplification primers (one pair) & lt spike gene The nucleotide sequence designed for PCR; 400 & gt; 2ggtctagagt tgtggtttca agtgattaggtaccaa tgccagtagt ggtg & lt; 210 & gt; 3

<211>50<212>DNA<213>人工序列<220> & Lt; 211 & gt; 50 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>根据spike基因碱基序列设计的用于PCR扩增spike基因M端的引物(一对)<400>3tttctagacc atgggttgtg tccttgctttggtacctc cgcctcgact tt<210>4<211>50<212>DNA<213>人工序列<220> & Lt; 223 & gt; amplification M terminal spike gene primers (one pair) & lt according spike gene nucleotide sequence designed for PCR; 400 & gt; 3tttctagacc atgggttgtg tccttgctttggtacctc cgcctcgact tt & lt; 210 & gt; 4 & lt; 211 & gt; 50 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>根据spike基因碱基序列设计的用于PCR扩增spike基因M端的引物(一对)<400>4tttctagacc atggcatata ggttcaatg ggttcaatg 4tttctagacc atggcatata; & lt; 223 & gt; spike gene amplification M end primers (one pair) & lt spike gene The nucleotide sequence designed for PCR; 400 & gt

ccggtaccat aagttcgttt atgtgt ccggtaccat aagttcgttt atgtgt

Claims (18)

1.一种SARS疫苗,其特征在于它包含SARS相关冠状病毒S基因和复制缺陷型腺病毒。 A SARS vaccine, characterized in that it comprises a SARS-associated coronavirus S genes and replication deficient adenovirus.
2.根据权利要求1所述的SARS疫苗,其特征在于缺陷型腺病毒为E1区完全缺失的C亚类的5型腺病毒。 2. SARS vaccine according to claim 1, characterized in that the C subclass deficient adenovirus E1 region is completely deleted adenovirus type 5.
3.根据权利要求2所述的SARS疫苗,其特征在于缺陷型腺病毒为E3区完全缺失的C亚类的5型腺病毒。 3. The SARS vaccine according to claim 2, characterized in that the C subclass deficient adenovirus E3 region is completely deleted adenovirus type 5.
4.根据权利要求2所述的SARS疫苗,其特征在于缺陷型腺病毒为E3区部分缺失的C亚类的5型腺病毒。 The SARS vaccine according to claim 2, characterized in that the defective adenovirus deleted for the E3 region portion C subclass of adenovirus type 5.
5.根据权利要求2所述的SARS疫苗,其特征在于缺陷型腺病毒为E3区不缺失的C亚类的5型腺病毒。 5. SARS vaccine according to claim 2, characterized in that the C subclass deficient adenovirus is not deleted E3 region of adenovirus type 5.
6.根据权利要求1或2所述的SARS疫苗,其特征在于缺陷型腺病毒内装CMV启动子和BGHpolyA。 6. SARS vaccine of claim 1 or claim 2, characterized in that the built-deficient adenovirus The CMV promoter and BGHpolyA.
7.根据权利要求1或2所述的SARS疫苗,其特征在于它包含SARS相关冠状病毒S基因全长。 SARS vaccine according to claim 1 or claim 2, characterized in that it comprises the SARS-associated coronavirus S full-length gene.
8.根据权利要求1或2所述的SARS疫苗,其特征在于克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S1结构域在内的序列。 SARS vaccine according to claim 1 or claim 2, wherein cloned into the adenoviral vector is a SARS-associated coronavirus S gene sequences include domains including S1.
9.根据权利要求1或2所述的SARS疫苗,其特征在于克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S2结构域在内的序列。 SARS vaccine according to claim 1 or claim 2, wherein cloned into the adenoviral vector is a SARS-associated coronavirus S gene sequences, including domains comprising S2.
10.根据权利要求1或2所述的SARS疫苗,其特征在于克隆到腺病毒载体中的为SARS相关冠状病毒S基因包括S1、S2结构域在内的序列。 SARS vaccine according to claim 1 or claim 2, wherein cloned into the adenoviral vector is a SARS-associated coronavirus S gene comprises a sequence S1, S2 including the domain.
11.根据权利要求1或2所述的SARS疫苗,其特征在于克隆到腺病毒载体中的为SARS相关冠状病毒S基因跨膜区(碱基号为:3686~3648,见附表一)及C端片段。 SARS vaccine according to claim 1 or claim 2, wherein the SARS-associated coronavirus was cloned into the S gene in the transmembrane region of the adenoviral vector (nucleotide numbers: 3686 ~ 3648, see Table I) and C-terminal fragment.
12.一种SARS疫苗的制备方法,包括(1)取得SARS相关冠状病毒的S基因;(2)将S基因与缺陷型腺病毒重组结合;(3)转染包装细胞;(4)经扩增、分离、纯化,制成制剂。 12. A method for the preparation of SARS vaccines, comprising (1) obtaining a SARS-associated coronavirus S gene; (2) combined with the S gene deficient recombinant adenovirus; (3) transfecting packaging cells; (4) the enlarged increase, isolated, purified, formulated.
13.根据权利要求12所述的SARS疫苗制备方法,其特征在于提取S基因后,将其克隆到pshuttle质粒,再将其与腺病毒骨架质粒(pAdeno-xTM)连接结合。 13. SARS vaccine production method according to claim 12, characterized in that after extraction of the S gene, cloned into the plasmid pshuttle, which is then connected to the adenoviral backbone plasmid (pAdeno-xTM) binding.
14.根据权利要求12所述的核酸疫苗的制备方法,其特征在于根据S基因序列设计PCR引物如下:v1 GGTCTAGAGT TGTGGTTTCA AGTGATv2 TTTCTAGACC ATGGGTTGTG TCCTTGCTV3 TTTCTAGACC ATGGCATATA GGTTCAATGV4 TAGGTACCAA TGCCAGTAGT GGTGV5 TTGGTACCTC CGCCTCGACT TTV6 CCGGTACCAT A AGTTCGTTT ATGTGT 12 14. The method of preparing the nucleic acid vaccine according to claim, characterized in that the S gene sequence according to PCR primers were designed as follows: v1 GGTCTAGAGT TGTGGTTTCA AGTGATv2 TTTCTAGACC ATGGGTTGTG TCCTTGCTV3 TTTCTAGACC ATGGCATATA GGTTCAATGV4 TAGGTACCAA TGCCAGTAGT GGTGV5 TTGGTACCTC CGCCTCGACT TTV6 CCGGTACCAT A AGTTCGTTT ATGTGT
15.根据权利要求12所述的SARS疫苗制备方法,其特征在于包装细胞为整合了C亚类5型腺病毒(Ad5)E1区基因的细胞系(株)。 15. The method of preparing SARS vaccine according to claim 12, wherein the packaging cell is a C alkylene integrated adenovirus type 5 (Ad5) E1 gene region of cell lines (strain).
16.根据权利要求15所述的SARS疫苗制备方法,其特征在于包装细胞为293细胞。 16. SARS vaccine production method according to claim 15, wherein the packaging cell is a 293 cell.
17.根据权利要求11所述的SARS疫苗制备方法,其特征在于所述制剂为喷雾剂或注射剂。 17. The method of preparing SARS vaccine according to claim 11, wherein said formulation is an injection or a spray.
18.SARS相关冠状病毒S基因在制备预防SARS的疫苗中的应用。 Application vaccine against SARS in 18.SARS associated coronavirus S gene in preparation.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079290A1 (en) * 2005-01-27 2006-08-03 Institute Of Laboratory Animal Science Of Chinese Academy Of Medical Sciences A recombinant sars-cov vaccine comprising attenuated vaccinia virus carriers
WO2007093133A1 (en) * 2006-02-16 2007-08-23 Chinese Center For Disease Control And Prevention Center For Aids/Std Control And Prevention Sars vaccine based on replicative vaccinia virus vector
CN100479858C (en) 2005-01-27 2009-04-22 中国医学科学院实验动物研究所;艾伦·戴蒙德艾滋病研究中心 A recombined smallpox vaccine - SARS vaccine and preparation method thereof
CN102844663A (en) * 2010-01-27 2012-12-26 俄勒冈健康科学大学 Cytomegalovirus-based immunogenic preparations

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079290A1 (en) * 2005-01-27 2006-08-03 Institute Of Laboratory Animal Science Of Chinese Academy Of Medical Sciences A recombinant sars-cov vaccine comprising attenuated vaccinia virus carriers
CN100479858C (en) 2005-01-27 2009-04-22 中国医学科学院实验动物研究所;艾伦·戴蒙德艾滋病研究中心 A recombined smallpox vaccine - SARS vaccine and preparation method thereof
WO2007093133A1 (en) * 2006-02-16 2007-08-23 Chinese Center For Disease Control And Prevention Center For Aids/Std Control And Prevention Sars vaccine based on replicative vaccinia virus vector
CN101020055B (en) * 2006-02-16 2012-08-08 中国疾病预防控制中心性病艾滋病预防控制中心 SARS vaccine based on replicative vaccinia virus vector
CN102844663A (en) * 2010-01-27 2012-12-26 俄勒冈健康科学大学 Cytomegalovirus-based immunogenic preparations
CN102844663B (en) * 2010-01-27 2016-01-06 俄勒冈健康科学大学 Based on the immunogenic formulation of cytomegalovirus

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