[Characterization of a recombinant Listeria monocytogenes strain containing the fusion protein gene of Newcastle disease virus].
Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P < 0.05). Such insertional mutation resulted in reduced virulence, about 3.7 logs and 6.5 logs less than its parent strain L. monocytogenes 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models respectively. The recombinant strain was relatively stable as shown by amplification of the target gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.
Xu
Jing-Jing
JJ
Zhejiang University Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory for Preventive Veterinary Medicine, Hangzhou 310029, China. brendajingjing@163.com
Jiang
Ling-Li
LL
Chen
Ning
N
Shuai
Jiang-Bing
JB
Fang
Wei-Huan
WH
D000818:Animals; D001422:Bacterial Adhesion; D004274:DNA, Recombinant / Q000235:genetics*; D005260:Female; D006367:HeLa Cells; D006461:Hemolysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008089:Listeria monocytogenes / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D009522:Newcastle disease virus / Q000235:genetics*; D009698:Nucleoproteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
D004274:DNA, Recombinant; D009698:Nucleoproteins; D014764:Viral Proteins