生长抑素(SS)与硫氧还原蛋白(Thioredoxin)相融合生成的2种基因工程化融合蛋白SS-N/X和SS-N/S的表达特性

Expressional Characteristics of the Two Engineered Fusion Proteins of Somatostatin (SS)-Thioredoxin:SS-N/X and SS-N/S

对基因工程化生长抑素 (som atostatin,SS) -硫氧还原蛋白 (Thioredoxin)融合蛋白 SS- N/ X和 SS- N/ S的表达产量、水溶性、Triton X- 10 0对 SS融合蛋白水溶性的影响、包涵体的溶解和复性特点以及 SS的抗原性与免疫原性等特性进行了研究。结果显示 ,在 30℃的低温下用 IPTG诱导表达 ,SS- N/ X融合蛋白主要以可溶性形式存在 ,占 75 .8% ,包涵体仅占 2 4 .2 % ;而 SS- N/ S融合蛋白则主要以包涵体的形式存在 ,占 81.1% ,可溶性蛋白仅占 18.9% ;32 C载体(p ET- 32 C)蛋白则居二者之间 ,可溶性蛋白与包涵体大约等比例出现。SS- N/ S在低温下诱导可超量表达 SS融合蛋白 ,其表达量约占菌体总蛋白的 4 0 .94 %。 0 .5 % Triton X- 10 0对融合蛋白的水溶性有非常显著的影响 ,几乎可使所有的 SS- N/ X融合蛋白和大部分 SS- N/ S融合蛋白及 32 C硫氧还原蛋白变成水溶性的。 32 C硫氧还原蛋白包涵体在尿素中的溶解度明显高于 SS- N/ S包涵体 ,2 mol/ L尿素可使近一半的 32 C硫氧还原蛋白包涵体溶解 ,而 SS- N/ S包涵体只有 2 7.5 %溶解 ;但在 5 mol/ L 尿素时 ,两者均可基本全部变为可溶性的。在 SS融合蛋白包涵体溶解时加入还原剂二硫苏糖醇 ,可明显降低 SS的抗原性。 SS融合蛋白

This research was conducted to investigate the characteristics of the two engineered fusion proteins of somatostatin (SS)-thioredoxin:SS-N/X and SS-N/S,including:expressional yields,solubility,Triton X-100 on the solubility of SS fusion proteins,the dissolving and renatured characteristics of inclusion bodies,and the antigenicity and immunogenicity of SS.The results showed:75.8% of the SS-N/X fusion protein was soluble,while only 18.9% of the SS-N/S fusion protein and approximately 50% of the 32C vector protein was soluble at 30℃;The SS-N/S fusion protein could be over-expressed when induced at lower temperature,with the highest yield reaching 40.94% of the total cell protein.The fusion proteins were very sensitive to 0.5% Triton X-100,almost all SS-N/X and most of SS-N/S fusion protein and 32C vector protein became soluble when inclusion bodies were washed with 0.5% Triton X-100.32C vector inclusion body has better solubility than SS-N/S in urea.Approximately half of the 32C vector inclusion body was redissolved in 2 mol/L urea,while only 27.5% of SS-N/S fusion protein was redissolved,but when increasing the concentration of the Urea to 5 mol/L,both of the proteins were almost redissolved.The SS antigenicity was obviously decreased when the inclusion body of SS-N/S fusion protein was redissolved in urea in the presence of DTT.These SS fusion proteins(soluble SS fusion proteins and renatured inclusion body) had higher SS antigenicity and immunogenicity.The SS vaccines were prepared by mixing the SS fusion proteins that were expressed in E.coli with equal Freund′s incomplete adjuvant.The growth rates of BALB/c mice,piglets and lambs improved significantly when immunized with the SS fusion protein vaccines.