tested with a 1 5 agarose gel electrophoresis and cut out of the gel under ultraviolet light the extracted fragments were purified by gel extraction kit and the resulted purified fragments were then used as the template for the second pcr reaction two sets of primers were used for pcr amplification of two heavy chain antibody vhh gene fragments the primers were designed as follows 3 3 restriction digestion of pcr product and construction of vhh antibody library 3 3 1 restriction digestion of pcr products and rphagemid pcantab 5e carrier the above pcr products and phagemid pcantab 5e carrier were digested by sfi i restriction enzyme after digestion by sfi i restriction enzyme the phagemid pcantab 5e vector and gene fragment were purified and quantified and ligation reaction was performed at a mass ratio of 1 3 in water at 16 u00b0 c for 14 hours the ligation product was transformed into e coli tg1 and 1 ul of transformed solution was plated 280 positive growing clones were obtained the following day 20 clones were randomly chosen for bacilli propagation and sequencing the results showed that 19 clones contained the construct sequence and most of the sequences were different it can be determined that vhh recombinant fragment insertion rate was about 95 the antibody library has good diversity it was calculated the vhh antibody library 39 s capacity was approximately 2 66 u00d7 105 cfu m13ko7 helper phage was inoculated in 2yt solid medium well separated plaques were chosen for propagation phage solution was diluted in 1 10 1 100 1 1000 and so on.