Abstract:
Objective To construct human single-chain variable fragment (scFv) antibodies gene library associated with esophageal cancer. Methods Metastatic periesophageal lymph nodes of esophageal cancer were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. Firstly, we screened gratefully two pairs of primers of the heavy and light regions separately, then the V
H and V
L fragments were first amplified from the cDNA. Secondly, the V
H-linker and V
L-linker were amplified from the V
H and V
L fragments. Lastly, SOE-PCR was used to connect the V
H-linker and V
L-linker to ScFv, the Sfi I and Not I restriction site was inlet in the scFv. The gel purified scFv gene repertoires are digested by Sfi I and Not I separately. The ligation mixes of scFv and pCANTAB-5E are transformed into competence E. coli TG1. The insert ratio of scFv antibodies library was identified by PCR. The products of Sfi I/ Not I double digestion reaction positive insert clone were identified by 1. 5 % agarose gel electrophoresis. Results Total RNA is good.The size of V
H is about 450bp, and V
L is about 350bp. The size of scFv is about 850bp. After transformation into E. coli TG1, 2 ×10
7 cfu/μg ampicillin resistant bacteria colonies grow after overnight culture. Randomly digestive reaction showed that the positive insert ratio was 91. 7 % (22/ 24) . Conclusion These results are bases to further phage antibody library construction.
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