ДНК-ДИАГНОСТИКА АНАПЛАЗМОЗА КРУПНОГО РОГАТОГО СКОТА

Objective of research: The purpose of our research was to develop a DNA diagnostic method for anaplasmosis in cattle. Materials and methods: Blood samples were obtained from the tail vein with the use of ethylenediaminetetraacetic acid as an anticoagulant. The extraction of DNA was performed with the kit Sorb-M. To analyze the gene msp4 the following sequences belonging to different isolates Anaplasma marginale were used. To select primers the conserved elements of sequences were detected with the server СlustalW2. The specificity of primers was checked by a BLASTN search. The results of polymerase chain reaction (PCR) were estimated using 2% agarose gel electrophoresis. Electrophoresis was performed within 40 minutes at the field intensity 5 V/cm. Fragments of gene msp4 obtained as a results of polymerase chain reaction (PCR) were purified, ligated and cloned in E. coli cells. Transformation was performed using the heat shock method. Search for E. coli colonies containing pGEM-msp4 plasmid was conducted by the PCR method using standard M13 primers with the following analysis of PCR results by electrophoresis. Target colonies of E. coli were cultured overnight at 37 °С in 2 ml LB medium containing ampicillin in a 100 mcg /ml concentration. Sequencing of received plasmids pGEM-msp4 was carried out by Sanger method and the genetic analyzer Applied Biosystems 3130.  Results and discussion: Development and approbation of primers on the basis of the MSP4 gene of Anaplasma marginale to perform DNA diagnostics of anaplasmosis in cattle by PCR method were described. Due to PCR sensitivity along with the use of primers, it is possible to identify 100 and more gene copies. TheЦель исследования – разработка метода ДНК-диагностики анаплазмоза крупного рогатого скота.  Материалы и методы. Пробы крови отбирали из хвостовой вены с использованием ЭДТА в качестве антикоагулянта. ДНК выделяли с помощью набора Sorb-M. Для анализа гена msp4 были использованы соответствующие последовательности, принадлежащие разным изолятам Anaplasma marginale. Выявление консервативных участков последовательностей для подбора праймеров проводили с помощью сервера СlustalW2. Видоспецифичность праймеров проверяли с использованием алгоритма BLASTN. Результаты полимеразной цепной реакции (ПЦР) оценивали методом электрофореза в 2%-ном агарозном геле. Электрофорез проводили в течение 40 минут при напряженности поля 5 В/см. Полученные в результате ПЦР фрагменты гена msp4 были очищены, лигированы и клонированы в клетках E. coli. Трансформацию проводили методом теплового шока. Поиск колоний E. coli DH5α, содержащих плазмиду pGEM-msp4, проводили методом ПЦР с использованием стандартных праймеров M13 с последующим анализом результатов ПЦР методом электрофореза. Целевые колонии наращивали в течение ночи при 37 °С в 2 мл среды LB, содержащей ампициллин в концентрации 100 мкг/мл. Секвенирование полученных плазмид pGEM-msp4 осуществляли по методу Сэнгера и генетического анализатора Applied Biosystems 3130. Результаты и обсуждение. Описаны разработка и апробация праймеров к гену msp4Anaplasma marginale для ДНК-диагностики анаплазмоза крупного рогатого скота методом ПЦР. Чувствительность ПЦР с использованием этих праймеров позволяет выявить 100 и больше копий гена. Проведенные испытания свидетельствуют о 100%-ной повторяемости и воспроизводимости данного метода

Spherical particles derived from TMV virions enhance the protective properties of the rabies vaccine

In this study the ability of spherical particles (SPs) obtained from the tobacco mosaic virus (TMV) virions to enhance the immunogenic potential of the vaccine was evaluated. TMV SPs were shown to increase the protective properties of the widely used effective Russian adjuvant-free rabies vaccine, composed of killed rabies virions. The results of the NIH potency test showed enhancement of protectivity, that is comparable with the effect of the incomplete Freund׳s adjuvant on the same vaccine. Keywords: Rabies vaccine, Plant virus, Spherical particles, Adjuvan

DNA DIAGNOSTICS OF ANAPLASMOSIS IN CATTLE

Objective of research: The purpose of our research was to develop a DNA diagnostic method for anaplasmosis in cattle. Materials and methods: Blood samples were obtained from the tail vein with the use of ethylenediaminetetraacetic acid as an anticoagulant. The extraction of DNA was performed with the kit Sorb-M. To analyze the gene msp4 the following sequences belonging to different isolates Anaplasma marginale were used. To select primers the conserved elements of sequences were detected with the server СlustalW2. The specificity of primers was checked by a BLASTN search. The results of polymerase chain reaction (PCR) were estimated using 2% agarose gel electrophoresis. Electrophoresis was performed within 40 minutes at the field intensity 5 V/cm. Fragments of gene msp4 obtained as a results of polymerase chain reaction (PCR) were purified, ligated and cloned in E. coli cells. Transformation was performed using the heat shock method. Search for E. coli colonies containing pGEM-msp4 plasmid was conducted by the PCR method using standard M13 primers with the following analysis of PCR results by electrophoresis. Target colonies of E. coli were cultured overnight at 37 °С in 2 ml LB medium containing ampicillin in a 100 mcg /ml concentration. Sequencing of received plasmids pGEM-msp4 was carried out by Sanger method and the genetic analyzer Applied Biosystems 3130.  Results and discussion: Development and approbation of primers on the basis of the MSP4 gene of Anaplasma marginale to perform DNA diagnostics of anaplasmosis in cattle by PCR method were described. Due to PCR sensitivity along with the use of primers, it is possible to identify 100 and more gene copies. Th