CONSTRUCTION OF INFECTIOUS FULL LENGTH CDNA CLONE OF DEN

DEN 感染性全长 CDNA 克隆的构建

• 批准号: 6545184
• 负责人: B FALGOUT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6545184
• 关键词: complementary DNA; dengue virus; electroporation; genetic manipulation; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein engineering

We are in the process of making full length "infectious" DEN1 cDNAs from both a virulent DEN1 virus, the Western Pacific (WP) strain, and from a live-attenuated vaccine candidate (PDK-20). The construction of the DEN1 WP infectious clone closely followed the methods used to make the infectious DEN2 clone. Briefly, the left-hand and right-hand ends of DEN1 Western Pacific strain were amplified by RT-PCR and then were cloned in proper orientation into the polylinker of the yeast shuttle vector pRS424. This DNA was linearized at a site in the polylinker between the two DEN ends. Meanwhile, a 5 kb cDNA was made from the middle of DEN1 by RT-PCR. This ~stuffer fragment~ overlaps both the left- and right-hand cDNAs contained in the pRS clone. Double homologous recombination between the stuffer fragment and the cloned left-hand and right-hand cDNAs upon co-transfection of yeast created a circular pRS plasmid containing the full-length DEN1 cDNA. Yeast colonies were screened by PCR, and plasmids containing full-length DEN1 cDNA were transfected into E. coli, DNA was prepared, and the structure confirmed by restriction analysis. Transcripts made from a series of independent clones produced typical dengue virus infections after electroporation into cells. Transcript-derived virus behaved like the parent WP virus in growth curves. The complete nucleotide sequence of one of the clones was determined, and the sequence of the WP RNA is about 75% completed; so far the two sequences are identical save for 1 silent mutation in the cloned cDNA. Work has begun to replace the FL DEN1 sequences with cDNA segments from the attenuated PDK-20 virus. First, an RT-PCR product representing the right half of PDK-20 was used to replace the same region of WP in the full-length clone. Transcripts from this chimeric construct were proven infectious. Then, the chimeric construct was used similarly to introduce the left half of PDK-20. Candidate clones have been identified and are being tested for infectivity. In addition, PDK-20 RNA is being sequenced. Once an infectious clone of PDK-20 is isolated, transcripts will be made and introduced into production Vero cells under cGMP by K. Eckels and his group at WRAIR. This candidate vaccine will then be compared in humans to the parental PDK-20 vaccine virus by B. Innis and colleagues at WRAIR. This will test whether an infectious clone of the average sequence of a virus population will behave the same in people as the parent vaccine virus. Having an infectious clone would allow the introduction ofspecific mutations into the PDK-20 virus in an effort to achieve further attenuation. Also, the Army group also has vaccine candidates for the other 3 dengue serotypes, and it is probable that at least two of these are not properly attenuated for use in humans. Thus, in the future, infectious clones could be derived from these other vaccine candidates and further mutagenized to achieve proper attenuation.

我们正在从一种强毒力的DEN1病毒西太平洋(WP)毒株和一种活的减毒候选疫苗(PDK-20)中制造全长的“传染性”DEN1 cDNA。DEN1可湿性粉剂感染性克隆的构建与DEN1感染性克隆的构建方法密切相关。简而言之，通过RT-PCR扩增了DEN1西太平洋株的左右两端，并将其定向克隆到酵母穿梭载体pRS424的多联体中。该DNA在两个末端之间的聚合体中的一个位置线性化。同时，通过RT-PCR从DEN1的中间片段中扩增出一条5kb的基因片段。这个~填充片段~与包含在PRS克隆中的左侧和右侧的cDNA重叠。酵母共转染后，填充物片段与克隆的左右两边的cDNA进行双同源重组，构建了包含全长DEN1基因的环状表达载体pRS。通过聚合酶链式反应对酵母菌落进行筛选，将含有全长DEN1基因的重组载体导入大肠杆菌，制备DNA，并进行限制性内切酶鉴定。由一系列独立克隆制成的转录本在电穿孔进入细胞后产生典型的登革热病毒感染。转录本来源的病毒在生长曲线上表现出与亲本WP病毒相似的行为。对其中一个克隆的核苷酸序列进行了测定，其中一个克隆的WP RNA测序完成了约75%；目前为止，这两个序列除了在克隆的cDNA中有1个无声突变外，其余都是相同的。用减毒的PDK-20病毒的cDNA片段替换FL DEN1序列的工作已经开始。首先，代表PDK-20右半部分的RT-PCR产物被用来替换全长克隆中相同的WP区域。这种嵌合结构的转录本被证明是有感染力的。然后，类似地使用嵌合结构来引入PDK-20的左半部分。候选克隆已经确定，并正在进行传染性测试。此外，PDK-20的RNA正在测序中。一旦分离出具有感染性的PDK-20克隆，K.Eockels和他在WRAIR的团队将在cGMP的作用下制作转录本，并将其导入生产的Vero细胞。这种候选疫苗随后将在人类身上与WRAIR的B.Inris和同事的亲本PDK-20疫苗病毒进行比较。这将测试病毒群体平均序列的感染性克隆是否会在人体内表现出与母体疫苗病毒相同的表现。拥有具有感染性的克隆将允许将特定的突变引入PDK-20病毒，以努力实现进一步的减毒。此外，陆军集团还拥有其他3种登革热血清型的候选疫苗，其中至少有两种可能没有适当地减毒用于人类。因此，在未来，感染性克隆可以从这些其他候选疫苗中衍生出来，并进一步诱变以实现适当的减毒效果。