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.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis:isolation and phylogenetic identification of the parasite

Background:Echinococcus multilocularis causes alveolar echinococcosis(AE)and is widely prevalent in Qinghai Province,China,where a number of different species have been identified as hosts.However,limited information is available on the Qinghai vole(Lasiopodomys fuscus),which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E.multilocularis.Thus,L.fuscus could contribute to the endemicity of AE in the area.Methods:Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E.multilocularis infection via anatomical examination.Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1.PCR-amplified segments were sequenced for a phylogenetic analysis.E.multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities.Results:Seventeen of the 50 Qinghai voles had E.multilocularis-infection-like vesicles in their livers.Eleven out of the 17 Qinghai voles presented E.multilocularis infection,which was detected by PCR and sequencing.The phylogenetic analysis showed that all 11 positive samples belonged to the E.multilocularis Asian genotype.A morphological identification and phylogenetic analysis of the E.multilocularis-infected Qinghai voles confirmed that all captured animals were L.fuscus.Conclusions:L.fuscus can be infected with E.multilocularis and plays a potential role in the life cycle and epidemiology of E.multilocularis in the Qinghai-Tibetan Plateau of China.