The Construction Of Human TRAb Phage Antibody Library

"My assignment is to construct a TRAb (TSH receptor antibody) combinatorial library phage antibody library from Graves patients.Methods: VL -linker- VH preparation. mRNA was extracted from peripheral blood lymphocyte (about 15 ml)of Graves patients who's TRAb and TSI were positive (detected by TRAb.TSI Assay Kit)using GffiCO' TRIZOL reagents. cDNA cloning was done by the Reverse Transcription System(Promega). Diverse libraries of immunoglobulin light (VL) chain variable gene was amplified using V K 5' primer which targets human first frame region and human V K 3' primer which targets J gene fragments while include 27 artificial oligonucleitides-liker. VH genes was the product of PCR using human VH5' linker which targets human first frame region while include 27 artificial oligonucleitides -liker ,and VH3' primer which targets joining fragments. We introduced four different digestion sites into these two pairs primers, facilitying subsequent digestion and cloning. Then, genes encoding single chain Fv fragments (ScFv)were made by randomly combing VH and VL genes by SOE (splicing overlap extension) PCR with a linker(artificial 36 oligonucleotides).The results: PCR products were electrophoresis on the 1.7%Agarose and VH and VL genes were prepared on i:he 420bp and 380bp respectively in accordance with other papers. Our primers are identified to be effective. We electrophoresis VL -linker- VH on the 1.7%Agarose and it can be seen on the 800bp, in accordance with what we expected. SOE method is proved that it can legate VH and VL genes by a linker.Next, the construction of the ScFv library. VL -linker- VH repertoire and p3SCMH were both digested and legated together, and the recombinants wereelectrotransformed into E.coli XLl-blue . I applied the method that combines the determination of the ratio of UV absorbance at 260 nm of DNA with ordinary fluorescent assay of DNA stained by ethidium bromide under UV irradiation to quantity DNA. During constructing a phage antibody repertoire, I tried to optimize several cross-linking factors (voltage, capacitor pulse time, etc) within a single experiment system. Samples were withdrawn for plating on SB medium dishes to determine Library size(Our experiment unit is individual ScFv recombinants).And our result is encouraging: a combinatorial library of 3.6X 106 members can meet subsequent requirements. Phagemid libraries were rescued after helper phage VCS-M13 was added and the supernatant culture was collected, centrifuged and re-suspended .The CPU (cloning forming units)wa4.2X 1014CFU/ml.The number is also confident.So we can get the conclusions below:l.The two primer pairs that target light and heavy chain variable can get single fragments. SOE is a less-time consuming, efficient way that can randomly legate PCR fragments.2.The most difficult work is how to get enough library size by electrotransformation. By optimizing relevant factors, increasing the quality of DNA and accurate DNA quantity, the desirable library size is finally reached, which is foundation for subsequent panning for TRAb.