Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity
Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity
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DOI：
10.1128/jvi.77.7.4357-4369.2003
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发表时间：
2003-04-01
影响因子：
5.4
通讯作者：
Enjuanes, L
中科院分区：
文献类型：
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作者：
Sola, I;Alonso, S;Enjuanes, L
冠状病毒传染性胃肠炎病毒（TGEV）的基因组已被改造为具有感染性cDNA的表达载体。该载体导致异源基因（绿色荧光蛋白[GFP]）的有效（>40 μ g/10（6）个细胞）和稳定（>20代）表达，其由插入先前由非必需ORF 3a和3b占据的位点中的开放阅读框（ORF）3a的转录调节序列（TRS）驱动。由该TRS驱动的表达水平高于在用N基因TRS工程化的调节序列控制下的表达盒的表达水平。包含GFP基因的重组TGEV仍然是肠致病性的，尽管肠组织生长减少了10- 10（2）倍。有趣的是，针对异源蛋白的特异性催乳免疫应答已在母猪及其后代中引起。在病毒基因N和7之间的异源基因的额外插入位点的工程化导致不稳定性和重组病毒3'端的新遗传组织。因此，从具有非典型核心序列5 '-CUAAAA-3'的TRS产生了主要种类的亚基因组mRNA。TRS和病毒前导序列3'端序列之间互补性的延伸与非典型核心序列的转录激活有关。工程载体导致的表达水平一样高的那些完善的载体，似乎非常有前途的疫苗的发展，并可能，基因治疗。
The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 mug/10(6) cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcription-regulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10- to 10(2)-fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3' end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5'-CUAAAA-3'. Extension of the complementarity between the TRS and sequences at the 3' end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.