Expression Of Spider Dragline Like Gene In Escherichia Coli And Milk Of Transgenic Mice

Spider dragline silk is an impressive material with a combination of tensile strength and elasticity that endows it with unique toughness and higher energy to break than any other common material, natural or artificial. Dragline silk is five times stronger by weight and lighter than steel. Because of its excellent mechanical properties the dragline silk could be useful for industrial, military, material, weave and medical purposes. This research focuses on acquiring artificial spider dragline silk protein gene which can express target protein efficiently and can be performed easily, and then produce spider silk in large quantities for industrial use.According to the known partial cDNA sequence of dragline silk, two artificial dragline silk gene monomers, a 360bp sequence and a 390bp sequence, were designed to encode analogs of the protein of Nephila clavipes dragline silk. DNA monomer sequences were multimerized to encode high molecular weight artificial spider dragline silk protein. Two-timer, four-timer, six-timer, eight-timer and sixteen-timer were respectively gained. According to the known partial amino acid sequence of dragline silk, an amino acid sequence of 19 AA was synthesized. Antiserum against dragline silk protein was raised in rabbits against synthetic peptides conjugated to keyhole limpet hemocyanin.Eight-timer and sixteen-timer of two artificial dragline silk gene monomers were cloned into pET-30a vector, a prokaryotic expression vector, and induced with IPTG. Four expression vector, pET30a-X8 pET30a-X16 pET30a-F8 and pET30a-F16 were constructed. Each strain produced a characteristic heterogeneous array of immunoreactive bands. The masses of the strongest band were respectively 85K.D 165KD 85KD and 165KD,which represents the full-length gene product in each pattern. The concentrations of recombinant dragline silk protein were respectively 800mg/L 500mg/L 200 mg/L and 200 mg/L.According to goat (i-casein signal sequence, a 125bp DNA sequence was designed and synthesized. Four-timer and six-timer of two artificial dragline silk gene monomers were ligated with the signal sequence and then were cloned into pBC1 vector, which is a specific milk expression vector. Four expression vector pBCl-F4, pBCl-X4, pBCl-F6 and pBCl-X6 were constructed.Transgenic mice were generated by microinjection of fertilized oocyte with two constructs, pBCl-F4 and pBCl-X6. For pBC1-X6 vector, transgenic mice were screened by PCR and Southern blot which revealed that 10 mice (5 5) among 58 mice were transgenic positive. The integration rate is 17%. F1 mice for most of founder line were also detected by PCR and the positive transgenic mice were found. Milk of five F0 mice and eight Fl mice was analyzed by Western blot and radioimmnoassay. Two F0 mice and seven Fl mice expressed recombinant dragline silk protein. The molecular masses of expressed recombinant dragline silk protein by most of mice were about 55KD, which was consistent with that of theoretic calculation. But some mice expressed recombinant dragline silk protein which molecular masses were not consistent with thatof theoretic calculation. In transgenic mice milk, the concentrations of reeombinant dragline silk protein were about 11 -50mg/L.For pBCl-F4 vector, transgenic mice were screened by PCR and Southern blot which revealed that 7 mice (4 3) among 59 mice were transgenic positive. The integration rate is 12%. F1 mice for each founder line were also detected by PCR and the positive transgenic mice were found. Milk of three FO mice and six Fl mice was analyzed by Western blot and radioimmnoassay. Two FO mice and two Fl mice expressed recombinant dragline silk protein. The molecular masses of expressed reeombinant dragline silk protein were about 40KD, which was consistent with that of theoretic calculation. In transgenic mice milk, the concentrations of reeombinant dragline silk protein were about 1 l-22mg/L.With artificial dragline silk gene being expressed in E. coli and transgenic mice milk, we found that our target gene construction was correct and...