Rescue of an Enterotropic Newcastle Disease Virus Strain ZM10 From Cloned cDNA and Stable Expressing an Inserted Foreign Gene

Background: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform.Results: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning (LIC) method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Conclusion: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry.

Serpins: Purification and characterization of potent protease inhibitors from Clostridium thermocellum

Clostridium thermocellum produces an extracellular cellulosome (a multiprotein complex produced by firmicutes bacteria), which, owing to its extracellular location, is open to protease attack. Serine protease inhibitors (serpins) protect bacteria against protease attack. However, their structure and function are poorly characterized. This study identified and amplified the serpin 1270 gene from the C. thermocellum genome. Purified serpins were cloned into the pTXB1 vector using the one-step sequence and ligation-independent cloning reaction and transformed into Escherichia coli BL21 DE3 cells. Enzyme overexpression and purification and enzyme inhibitory assays were performed. The results showed that serpin 1270 has 89% inhibition against Bacillus subtilisin and 64% inhibition against trypsin, chymotrypsin, and papain.

Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System

The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. The completed genome sequence of V. dahliae has facilitated large-scale investigations of individual gene functions using gene-disruption strategies based on Agrobacterium tumefaciens-mediated transformation. However, the construction of gene-deletion vectors and screening of deletion mutants have remained challenging in V. dahliae. In this study, we developed a fast and easy gene knockout system for V. dahliae using ligation-independent cloning and fluorescent screening. We identified secretory factor VdASP F2 in a T-DNA insertion library of V. dahliae and deleted the VdASP F2 gene using the developed knockout system. Phenotypic analysis suggests that VdASP F2 is not necessary for V. dahliae growth on potato dextrose agar under various stress conditions. However, on semisynthetic medium or under limited nutrient conditions at lower temperatures, the VdASP F2 deletion mutant exhibited vigorous mycelium growth, less branching, and a significant delay in melanized microsclerotial formation. Further assessment revealed that VdASP F2 was required for the expression of VDH1 and VMK1, two genes involved in microsclerotial formation. Cotton inoculated with the VdASP F2 deletion mutant wilted, demonstrating that VdASP F2 is not associated with pathogenicity under normal conditions. However, after inducing microsclerotial formation and incubation at low temperatures, cotton infected with the VdASP F2 deletion mutant did not exhibit wilt symptoms. In conclusion, our results show that VdASP F2 plays an important role in the response of V. dahliae to adverse environmental conditions and is involved in a transition to a dormant form for prolonged survival.