Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393
Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393
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猪轮状病毒外衣壳蛋白VP4在干酪乳杆菌ATCC393中的定向染色体整合和表达
DOI：
10.1007/s00253-016-7779-y
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发表时间：
2016-08
影响因子：
5
通讯作者：
Li, Yi-Jing
中科院分区：
文献类型：
--
作者：
Bukhari, Syed M.;Tang, Li-Jie;Xu, Yi-Gang;Li, Yi-Jing
在5-氟尿嘧啶（5-FU）存在下，使用两步质粒整合，我们开发了一种稳定且无标记的干酪乳杆菌菌株用于疫苗抗原表达。L的upp。使用编码尿嘧啶磷酸核糖基转移酶（UPRTase）的干酪素作为反选择标记。我们采用对5-FU具有抗性的Δupp等基因突变体作为宿主，并使用带有upp表达盒的温度敏感性自杀质粒作为反向选择整合载体。UPRTase的染色体外表达补充了突变的染色体upp等位基因，并恢复了对5-FU的敏感性。所得基因型可以是野生型或重组型。通过插入和表达猪轮状病毒（PRV）VP 4证明了该系统的有效性。为了提高VP 4的表达，我们分析了L. caseI转录谱，并选择组成型高表达烯醇化酶基因（eno）。在5-FU存在下筛选插入eno终止密码子后的VP 4。利用基因组PCR扩增，我们证实VP 4成功整合并稳定遗传至少50代。Western blot结果表明，VP 4在不同糖份的培养基中均能稳定表达。RT-qPCR和ELISA分析表明，VP 4从染色体位置的表达是类似的质粒表达系统实现的。将重组菌株经口免疫BALB/c小鼠，结果表明，表达VP 4的L.酪蛋白可诱导小鼠产生特异性的局部和全身体液免疫反应。总之，改进的基因置换系统是一种有效的染色体重组方法。并为疫苗生产提供了一种安全的工具。
Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.
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DOI：
--
发表时间：
2007-03
期刊：
Sheng wu gong cheng xue bao = Chinese journal of biotechnology
影响因子：
--
作者：
Yigang Xu;Li-chun Cui;Guang-peng Ma;Lijie Tang;Jun-wei Ge;Chun-li Xia;X. Qiao;Li-li Zhao;Yi-jing Li
通讯作者：
Yigang Xu;Li-chun Cui;Guang-peng Ma;Lijie Tang;Jun-wei Ge;Chun-li Xia;X. Qiao;Li-li Zhao;Yi-jing Li
作为抗传染性胃肠炎病毒口服干酪乳杆菌疫苗的佐剂，上调 Th1 和 Th17 细胞中的 MDP 和 tuftsin 基因表达
影响因子：
5
作者：
Jiang, Xinpeng;Yu, Meiling;Li, Yijing
通讯作者：
Li, Yijing
影响因子：
14.9
作者：
P. Pouwels;J. Leunissen
通讯作者：
P. Pouwels;J. Leunissen
影响因子：
5.4
作者：
Erich R. Mackow;P. Vo;R. Broome;Dorsey Bass;Harry B. Greenberg
通讯作者：
Erich R. Mackow;P. Vo;R. Broome;Dorsey Bass;Harry B. Greenberg
影响因子：
5.4
作者：
P. Offit;R. Shaw;H. Greenberg
通讯作者：
P. Offit;R. Shaw;H. Greenberg