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将猪胰岛素前体（ Ｐ Ｉ Ｐ） 基因和在其５′端引入９ 个氨基酸的间隔肽序列的 Ｐ Ｉ Ｐ 基因（ｓｐ Ｐ Ｉ Ｐ） 插入到 Ｐｉｃｈｉａｐａｓｔｏｒｉｓ 的分泌表达质粒ｐ Ｐ Ｉ Ｃ９ 中， 得到分泌表达质粒ｐ Ｐ Ｉ Ｃ９／ Ｐ Ｉ Ｐ 和ｐ Ｐ Ｉ Ｃ９／ｓｐ Ｐ Ｉ Ｐ 并用以转化 Ｐ．ｐａｓｔｏｒｉｓ Ｇ Ｓ１１５ 。用点杂交筛选， 获得高拷贝转化子 Ｐ３９（ｓｐ） 和 Ｓ５１（ ＋ ｓｐ） 。经１ Ｌ 摇瓶培养， Ｐ３９ 和 Ｓ５１ 在 Ｐ．ｐａｓｔｏｒｉｓ 中分泌表达 Ｐ Ｉ Ｐ 和ｓｐ Ｐ Ｉ Ｐ 的量分别为１０ ｍｇ／ Ｌ 和４０ ｍｇ／ Ｌ。ｓｐ Ｐ Ｉ Ｐ 在 Ｐ．ｐａｓｔｏｒｉｓ 中的表达水平高于其在本实验室报道的 Ｓ．ｃｅｒｅｖｉｓｉａｅ 和 Ｋ．ｌａｃｔｉｓ 中的表达， 表明间隔肽的引入有助于提高 Ｐ Ｉ Ｐ 的表达。用１０ Ｌ 罐发酵ｓｐ Ｐ Ｉ Ｐ 表达量达２５０ ｍｇ／ Ｌ。 Ｐ Ｉ Ｐ 和ｓｐ Ｐ Ｉ Ｐ 经转肽得到重组人胰岛素， 其受体结合能力与猪胰岛素相同， 体内生物活力２７ Ｉ Ｕ／ｍｇ 。
The porcine insulin precursor (P I P) gene and the P I P gene (sp PIP) which introduced a 9-amino acid spacer peptide sequence at its 5 ’end were inserted into the secretory expression plasmid p P I C9 of Pichia pastoris, The secretory expression plasmids p P I C9 / P I P and p P I C9 / sp P I P were obtained and used to transform P. pastoris G S115. Screening with point hybridization, access to high copy transformants P39 ( sp) and S51 (+ sp). After 1 L shake flask culture, P39 and S51 in P. The amounts of PIP and sp-PIP secreted by pastoris were 10 mg / L and 40 mg / L, respectively. sp P P I P The expression level in pastoris is higher than that reported in our laboratory. cerevisiae and K. lactis, suggesting that the introduction of a spacer peptide can help improve the expression of PIP. With 10 L tank fermentation sp P I P expression of 250 mg / L. P I P and sp P P by recombinant peptide to obtain recombinant human insulin, its receptor binding capacity and insulin in pigs the same, in vivo biological activity 27 I U / mg.