Global Expression Profiling Of M.Bovis Under The Zinc And Iron Deprivation Condition And PHOP Gene Deletion Vector Construction

Bovine tuberculosis (bTB) is a chronic infectious zoonotic disease that shares between livestock and human beings and there is no effective and rapid control method so far. A large amount of cattle herds with high M.bovis infection rates was documented worldwide and wildlife reservoirs constitute a source for re-infection of cattle in many countries either. The control and eradication of this disease are mainly based on the test and slaughter policy. Various factors, such as high cost and wildlife reservoirs, have been identified as the major obstacles to this eradication plan. Many developed countries focus on bTB vaccine development in order to make a good progress in the bTB control. Current attempts to develop an effective bTB vaccine are based on two approaches:boosting BCG or attenuated M.bovis strain. Both BCG and rBCG vaccines were evaluated in cattle. But the results of these tests showed that high variable efficacy in protection in cattle. Previous works showed the attenuated live tuberculosis vaccine SO2 based on phop mutant has become one of the candidates in clinical trials, which shows a good perspective for bTB vaccine development.Based on the above viewpoints, an allelic exchange method was employed to generate a gene deletion mutant of M.bovis in this study. Moreover, the whole genome mycobacterium tuberculosis microarray was used to investigate the different gene expression between M.bovis strain and BCG strain under the iron deprivation conditions and the zinc deprivation conditions.1. Iron deprivation condition affected the bacterium growthGrowth curve of BCG at iron deprivation condition showed that the time of exponential growth phase became shorter and the biomass reduction reflected by OD600 value was observed during the Stationary-Phase.2. Differential gene expression between M.bovis and BCG strain under the iron deprivation condition and the zinc deprivation conditionAgilent whole genome mycobacterium tuberculosis microarray was used to screen the gene expression differences between M.bovis strain and BCG strain. Following 24 hours of incubation under the different conditions: CMM, CMM(△Fe△Zn), CMM(△Zn) and CMM(△Fe), total RNA of those samples were extracted for synthesis of fluorescently labeled cDNA (Cyanine-3 for samples from M.bovis,Cyanine-5 for samples from BCG). A total 4842 genes were tested by the whole genome microarray. Compared to those in the condition of CMM,236 genes were identified to be differentially expressed in the conditions of CMM(△Fe△Zn), CMM(AZn) and CMM(△Fe). These genes encode a range of functions, including transcriptional regulators, enzymes, cell wall and secreted proteins, PE/PPE proteins, lipid metabolism and hypothetical proteins. A heatmap was used to classify the 236 genes to give us a further insight into understanding the gene coding hypothetical protein. For three genes (Rv3312A,Rv2926c,Rv1993c) tested, the RT-qPCR results corroborated the microarray data.3. Construct the suicide vector for gene deletion mutant of M.bovisTo construct the mutant,632bp upstream region of phop and 587bp downstream region of phop were amplified by PCR. A marker gene cassette containing three selective marker genes (LacZ, SacB, Hygr) was digested from the pGOAL plasmid. The two regions of phop and the marker gene cassette were inserted into the P2NIL vector to generate the final suicide vector. After verifying the functions of the three selective marker genes in E.coli, the suicide vector plasmid DNA was electroporated into M.bovis to generate the crossing over events. So far, no recombinant was observed on the selective plate.