Intracellular And Secretory Expression Of Common Carp Growth Hormone CDNA In The Yeast Pichia Pastoris

Growth hormone is a polypeptide produced by the pituitary cells to regulate growth and metabolisms in vertebrates. Of-I has been shown to enhance the appetite, feed conversion efficiency and growth rate of fish, Therefore, it was said that OH may be the most promising agent for growth promoting in aquaculture. However, the content of native Gil in pituitary is extremely low and its availability is limited. Fortunately, the recombinant fish OHs which proved in many cases the same function as the native ones, can be produced in abundance. The yeast Pichia pczstoris is a novel host for producing high levels of functionally active recombinant proteins. In this research, recombinant carp (Cypritws cat plo) Gil (rcGH) were produced both in intracellular and secretory manner. 1. Intracellular expression of cGH cGH eDNA was cloned into the yeast Pichia paslOri.S?integrative expression vector pHiL -D2, under the control of the alcohol oxidase promotor. The Iineanized recombinant plasmid was then transformed into l-11S4 mutant yeast strain OS 115 by electroporation. Genomic DNA was detected to screen the positive transformants. Mut8 and Mutt clones were separated on MM medium. After methanol inducement, the cell was collected and disrupted by sonicator. The supernatant of cell Iysates was examined on SDS-PAGE gel. A specific protein band about 22kD appeared with the same molecular weight as the cGH marker, accounting for about 2-3%. Western blotting was performed to further identify it to be rcGH. By oral administration as feed supplement, rcGH showed significant growth romoting effect in weight on tilapia fingerling. 2. Secretory expression of cGI-i cGH eDNA was cloned into the secretion vector pPJCZx-A, following a cr-factor signal sequence. GS 115 was transformed by the recombinant vector. Zeocin was used to screen the recombinant yeast clones. After inducement by methanol, the supernatant was detected by SDS-PAGE. Results show that rcGI-I was secreted into the medium, with the highest production of cGH at 72hr on inducement. Expression in different pH (4.5, 5.5, 6.0, 6.5 ) values were also conducted, results revealed that the optimum was 6.0. Analyzed with the Tiger Gel Image Scanner, secreted rcGH accounted for 36.41% of the total supernatant proteins with the yield of 300-400mg/L. The ferment supernatant was separated through Qepharose anion xchange chromatography. The purified and lyophilized rcGH was dissolved in the sterile saline and was injected tilapia intraperitoneally at the dose of 0.lug/g body weight for one tested group, and 1 ug/g body weight for another. Control group is treated with sterile saline without rcG VI. After 5 injections, The rcGH-treated groups showed improved growth rate compared with the control one.