Cattle Peripheral Blood Mononuclear Cells Yeast Double Cross Cdna Library Build And Identification

Brucellosis is endemic in livestock an humans in Uganda and its transmission involves a multitude of risk factors. Brucellosis in cattle is almost exclusively caused by B.abortus. Brucellosis biovars and genotypes are known to be regionally restricted in their distribution. We constructed the cDNA library of the PBMC. We will find the cell receptor in the library with yeast two-hybrid. It is very effective for Brucellosis prevention and manipulation.In the research, first we make the PBMC, then extract the total RNA by using Trizol method. And we constructed the yeast two-hybird cDNA library of the PBMC with the method of SMART. The procedures including: the first strand synthesis, amplification of ds cDNA by long distant PCR. Using column purification of ds cDNA with a CHROMA SPIN TE-400 column, preparation of Competent yeast cells. Transformation of the Competent Yeast Cells with ds cDNA and PGADT7-Rec, two-hybrid library screened and identified. Library construction takes place directly in our library yeast strain Y187. Then screen the positive colonies on SD/Leu plates, calculate the titer and the transformation efficiency. Finally we examined how long the inserted of the ds cDNA by colony PCR.The examined result of the total RNA of the PBMC by ultraciolet spectrophotometer and 1% agarose gel electrophoresis OD260/OD280=1.95,28s and 18s was clearly. So the concentration and purifty was food, meet the requirement of constructing cDNA library. The fragment of the purified ds cDNA was 400-2500bp. And the transformation efficiency of the cDNA library was 4.0×108 CFU/mL. And the recombination fraction was above 95%.And the average fragment of the inserted cDNA was 700bp.All the test result indicated that the quality of the cDNA library was very good, and it can be used for yeast two-hybrid. It also provided important materials and basement for screening the Brucellosis.