Different Strategies for Preparation of Non-tagged rV270 Protein and Its Efficacy against Yersinia Pestis Challenge

Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Identification of Legionella species by the composition of cellular fatty acids

One hundred andfifty-three isolates from the environment and 36 reference strains of the Legionella were studied with regards to their composition of cellular fatty acids as determined by gas chromatography,and then were classified into 41 groups by numerical analysis.Most reference strains formed only a single group,except L.micdadei,L.jamestowniensis,L.parisiensis,L.jorda-nis,L.feeleii and L.longbeachae,which were clustered into two or three groups.Even serological types of L.pneumophila could be clearly identified.Therefore,in this study,numerical analysis of cellular fatty acid composition is an effective method for identifying Legionella species.

In silico assessment of the impact of 2019 novel coronavirus genomic variation on the efficiency of published real-time quantitative polymerase chain reaction detection assays

To the Editor:In December 2019,coronavirus disease 2019 outbreak caused by the 2019 novel coronavirus(2019-nCoV)happened in Wuhan,China.Now,it has posed a worldwide public health threat.Real-time quanti-tative polymerase chain reaction(RT-qPCR)was recom-mended as an effective pathogen detection method and has played an important role in prevention and control of the current outbreak.Many research institutions have released their primer sets for RT-qPCR.If the variant sites were located in the primer regions,the efficiency of RT-qPCR would be reduced,thus possibly causing false negative results,and leading to unpredictable impact on the diagnosis of patients and the control of this outbreak.Therefore,a comprehensive investigation on 2019-nCoV genome variation is necessary to evaluate the effectiveness of current released RT-qPCR methods.