纯化重组霍乱毒素B亚单位与天然霍乱毒素B亚单位性质的对比研究

霍乱毒素Ｂ亚单位（ＢＳ）已用于新型口服霍乱疫苗、佐剂及蛋白质载体，但成本高，来源困难．用重组霍乱毒素Ｂ亚单位（ｒＢＳ）代替ＢＳ可克服上述缺点．ｒＢＳ用于上述目的前必须证实其在物理、化学及免疫学性质方面与天然同类产品的一致性．用亲和层析法从各批次大罐发酵所获工程菌Ｅ．ｃｏｌｉＭＭ２（ｐＭＭ－ＣＴＢ）培养物上清中制备得到了小批量ｒＢＳ纯品，在同等条件下与ＢＳ（Ｓｉｇ－ｍａ公司产品）进行理化、免疫学性质的对比研究，证实二者在ＳＤＳ－ＰＡＧＥ中电泳带位置一致、分子量相同，纯度达９９％；在反相ＨＰＬＣ中出峰行为一致，纯度达１００％；在半干式聚焦电泳分析中电泳带分布相同，等电点为７．９１．ｒＢＳＮ端起的２０个氨基酸序列为ＴＰＱＮＩＴＤＬＣＡＥＹＨＮＴＱＩＨＴＬ，与克隆基因来源株的毒素Ｂ亚单位同一段序列完全一致．氨基酸组成分析证实ｒＢＳ与ＢＳ相近．在免疫学性质分析中，ｒＢＳ与ＢＳ在免疫双扩散试验中与抗ＣＴ均出一条沉淀线且相互吻合；在免疫电泳试验中二者与抗ＣＴ在相应位置上产生一条沉淀弧；二者均能与神经节苷脂ＧＭ１结合且这种结合均可通过二者与抗ＣＴ的预保温处理而被阻断．对比研究结果揭示ｒＢＳ与ＢＳ性质完全一致，可代替ＢＳ用于

Studies on the Characters of the Recombinant Cholera Toxin B Subunit in Comparison with the Native Cholera Toxin B Subunit

An engineered bacterium strain E.coli MM2(pMM CTB)which secrete cholera toxin B subunit at high level has been constructed by recombinant DNA technigue.It was confirmed that the strain was maintained to secrete the recombinant cholera toxin B subunit(rBS)as original by fermentation in 50 L fermentor(type MSJ U3 made in Japan).In order to use the rBS as a novel type of cholera vaccine,adjuvant and protein carrier,its characters must be checked against those of native cholera toxin B subunit(BS)in chemistry and physics and immunity.The pure rBS was prepared by affinity chromatography from culture supernatant of E.coli MM2,which was obtained in a few batches of products by fermentation.The characters of purified rBS were checked under similar conditions against those of native BS purchased from Sigma.Both rBS and BS exhibited one protein band by SDS PAGE,and were in the same position on polyacrylamide gel with M r 11 677.The purity of purified rBS was 98 76%(stained by Coomassie blue)or 99 16%(silver staining).The peak behavior of rBS in HPLC was identical with that of the native B subunit.The purity of rBS in HPLC was 100%.Analysis of the 20 amino acid residues from N terminal of rBS was TPQNITDLCAEYHNTQIHTL which was homologous with that of the natural B subunit of Vibrio cholerae 569B.The amino acid composition analysis of the rBS showed no deferences with that of the native B subunit.The contents of a few of amino acids were lower than theoretical value due to their instability or destruction in hydrolysis under acid condition.No rise in the values was detected.Antigenicity studies indicated that the engineered B subunit gave a single precipitation line which connected the line formed by the natural B subunit.The same finding was observed in the microimmunoelectrophoresis.The rBS could bind the receptor ganglioside GM1 in GM1 ELISA and this binding could be blocked by preincubation of rBS with antiserum angainst cholera toxin.In summary,It is demonstrated that the characters of rBS are identical with those of native BS in physics and chemistry and immunity,thus,the rBS can be used in vaccine and as adjuvant and protein carrier to replace BS.