提高破伤风类毒素免疫原性的初步研究
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摘要
破伤风梭菌产生的破伤风毒素,经0.4%甲醛灭活后,失去毒性而仍保留抗原性称之为破伤风类毒素。类毒素精制后,可作为疫苗对人和动物进行免疫接种。但机体感染后不能获得终身免疫力,且此免疫程序较长。为了减轻接种的副反应,提高免疫效果,减少注射次数,提高接种覆盖率,本研究采用原核细胞表达具有高免疫原性的重组C蛋白,用于生产疫苗及免疫接种,以避免出现副作用;另外,用双功能交联剂戊二醛聚合破伤风毒素,使毒素分子之间进行交联,从而产生分子量较大的,表面决定簇多的聚合类毒素。该类毒素作为免疫的抗原,可以诱发较高的抗体水平,加强免疫记忆,延长保护机体免受破伤风的侵害的时间。
1.利用PCR技术从破伤风梭菌基因组DNA中扩增出1 356 bp的破伤风毒素C基因,将该基因片段插入到表达载体pET32a(+)中,转化宿主菌BL21(DE3)中,用IPTG诱导表达,观察各种条件下对重组菌蛋白表达的影响。对重组后蛋白用Ni-NAT亲和层析的技术纯化C融合蛋白,然后进行小鼠免疫实验并测定效价。研究显示:经SDS-PAGE分析,重组破伤风毒素C蛋白(rTTC)的表达量占可溶性蛋白的28.19%,纯化后得到蛋白纯度为96.92%。免疫印迹证实该重组蛋白是破伤风毒素C蛋白抗原,对小鼠免疫,三免后14 d rTTC组小鼠抗体效价仍能达到1∶16 000。所获得的重组蛋白具有良好的免疫原性。
2.经过厌氧培养的破伤风梭菌,产毒后进行脱毒、精制,在产毒期间进行毒力鉴定、解毒和毒性逆转实验。再由戊二醛聚合破伤风类毒素,经不同的聚合条件的优化,进行分子量和Lf的测定,然后免疫小鼠和兔子,并测定其免疫效价。培养的毒素可以作为制备类毒素使用,脱毒精制后的类毒素,经毒力鉴定试验、解毒试验和毒性逆转试验检测后均合格。用pH 7.2的PBS稀释类毒素,终浓度为0.1%、0.3%戊二醛置于室温中聚合稀释后的类毒素12 h,聚合类毒素中聚合的类毒素分子量为600 ku,含量占聚合物的43.82%,经ELISA检测抗原性,抗原与阳性血清呈阳性反应。免疫小鼠和兔子,平均效价在1∶2 0 000～1∶3 0 000。所得到的聚合类毒素具有抗原性及免疫原性。
3.最后将聚合类毒素和未聚合类毒素分别与重组抗原联合免疫马匹,未聚合类毒素的平均效价明显低于聚合类毒素组别,说明在联合免疫后,聚合类毒素可以提高马匹的血清效价。
Tetanus toxoid (TT) consists of a formaldehyde-treated tetanus toxin is produced by Clostridium tetanus under anaerobic conditions, and does not have the toxicity, but still contains the antigenicity. After refined, it can be used as vaccine for tetanus prophylaxis. The disease itself does not induce immunity. Therefore, prophylaxis against tetanus is usually conducted by frequent boosters throughout the life of human and animals. To relieve side-effects, boost the result of immunization, reduce injection times, raise the coverage area of immunity against the toxin, the experiment used the prokaryocyte vector to express the C fragment of tetanus toxin which has high immunogenicity. The recombinant C fragment of tetanus toxin (rTTC) was expressed to produce vaccine and used to immunize human and animals in order to avoid severe adverse reactions. In the other hand, the difunctionality cross-linking agent polymerized toxin can conjugate toxin to polymeride between toxin molecules. The polymeride toxoid has many epitopes and large molecular weight, and can be used as antigen to stimulate high antibody level and protect the human and animals to avoid tetanus toxin attack.
The fragment C gene of tetanus toxin was amplified from Clostridium tetani genomic DNA by PCR. It was inserted into the high-expression vector pET32a, and expression of this plasmid in Escherichia coli BL21 (DE3) resulted in the production of a fusion protein. The expression was induced by IPTG and the fusion protein expression was observed under different conditions. The fusion protein was purified significantly in one step by affinity chromatography then used to immunize mice, and the antibody titers were tested. The result of SDS-PAGE showed that a specific recombinant protein was expressed and accounted for 28.19% of the soluble protein. The final purity was 96.92%. After the third immunization 14 days, the anti-tetanus antibody titers of the mice in rTTC group were detected. The recombinant protein is an immunogenic antigen of tetanus toxin and thus constructs a basis for the current vaccine in the future.
The tetanus toxin were produced by cultured C. tetanus in a strict anaerobiosis condition, were removed the toxigenicity, and refined. During that time, qualification of toxigenicity
1.利用PCR技术从破伤风梭菌基因组DNA中扩增出1 356 bp的破伤风毒素C基因,将该基因片段插入到表达载体pET32a(+)中,转化宿主菌BL21(DE3)中,用IPTG诱导表达,观察各种条件下对重组菌蛋白表达的影响。对重组后蛋白用Ni-NAT亲和层析的技术纯化C融合蛋白,然后进行小鼠免疫实验并测定效价。研究显示:经SDS-PAGE分析,重组破伤风毒素C蛋白(rTTC)的表达量占可溶性蛋白的28.19%,纯化后得到蛋白纯度为96.92%。免疫印迹证实该重组蛋白是破伤风毒素C蛋白抗原,对小鼠免疫,三免后14 d rTTC组小鼠抗体效价仍能达到1∶16 000。所获得的重组蛋白具有良好的免疫原性。
2.经过厌氧培养的破伤风梭菌,产毒后进行脱毒、精制,在产毒期间进行毒力鉴定、解毒和毒性逆转实验。再由戊二醛聚合破伤风类毒素,经不同的聚合条件的优化,进行分子量和Lf的测定,然后免疫小鼠和兔子,并测定其免疫效价。培养的毒素可以作为制备类毒素使用,脱毒精制后的类毒素,经毒力鉴定试验、解毒试验和毒性逆转试验检测后均合格。用pH 7.2的PBS稀释类毒素,终浓度为0.1%、0.3%戊二醛置于室温中聚合稀释后的类毒素12 h,聚合类毒素中聚合的类毒素分子量为600 ku,含量占聚合物的43.82%,经ELISA检测抗原性,抗原与阳性血清呈阳性反应。免疫小鼠和兔子,平均效价在1∶2 0 000～1∶3 0 000。所得到的聚合类毒素具有抗原性及免疫原性。
3.最后将聚合类毒素和未聚合类毒素分别与重组抗原联合免疫马匹,未聚合类毒素的平均效价明显低于聚合类毒素组别,说明在联合免疫后,聚合类毒素可以提高马匹的血清效价。
Tetanus toxoid (TT) consists of a formaldehyde-treated tetanus toxin is produced by Clostridium tetanus under anaerobic conditions, and does not have the toxicity, but still contains the antigenicity. After refined, it can be used as vaccine for tetanus prophylaxis. The disease itself does not induce immunity. Therefore, prophylaxis against tetanus is usually conducted by frequent boosters throughout the life of human and animals. To relieve side-effects, boost the result of immunization, reduce injection times, raise the coverage area of immunity against the toxin, the experiment used the prokaryocyte vector to express the C fragment of tetanus toxin which has high immunogenicity. The recombinant C fragment of tetanus toxin (rTTC) was expressed to produce vaccine and used to immunize human and animals in order to avoid severe adverse reactions. In the other hand, the difunctionality cross-linking agent polymerized toxin can conjugate toxin to polymeride between toxin molecules. The polymeride toxoid has many epitopes and large molecular weight, and can be used as antigen to stimulate high antibody level and protect the human and animals to avoid tetanus toxin attack.
The fragment C gene of tetanus toxin was amplified from Clostridium tetani genomic DNA by PCR. It was inserted into the high-expression vector pET32a, and expression of this plasmid in Escherichia coli BL21 (DE3) resulted in the production of a fusion protein. The expression was induced by IPTG and the fusion protein expression was observed under different conditions. The fusion protein was purified significantly in one step by affinity chromatography then used to immunize mice, and the antibody titers were tested. The result of SDS-PAGE showed that a specific recombinant protein was expressed and accounted for 28.19% of the soluble protein. The final purity was 96.92%. After the third immunization 14 days, the anti-tetanus antibody titers of the mice in rTTC group were detected. The recombinant protein is an immunogenic antigen of tetanus toxin and thus constructs a basis for the current vaccine in the future.
The tetanus toxin were produced by cultured C. tetanus in a strict anaerobiosis condition, were removed the toxigenicity, and refined. During that time, qualification of toxigenicity
引文
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