Expression of full and fragment-B of diphtheria toxin genes in Escherichia coli for generating of recombinant diphtheria vaccines

Shaimaa Abulmagd, A. E. Khattab, H. Zedan
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Abstract

Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.
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白喉毒素全片段B基因在大肠杆菌中的表达及重组白喉疫苗的制备
目的本研究将白喉棒状杆菌Park William的白喉毒素(dt)和B片段(dtb)基因克隆到大肠杆菌中,并对纯化的表达蛋白进行评价,最终用作候选疫苗。材料和方法从ATCC(American Type Culture Collection)13812菌株中分离出dt和dtb基因。质粒pET29a+通过DNA spin-TM质粒纯化试剂盒提取,其中使用BamHI和HindIII-HF插入基因。将克隆的pET29a+dt和pET29a+dtb质粒转化到大肠杆菌BL21(DE3)PlysS中作为表达宿主。通过对NCBI(国家生物技术信息中心)GenBank中所有报道的核苷酸序列爆破序列(BLASTn)来验证序列的同一性。通过不同类型和参数的发酵来确定高产蛋白质的最佳条件。最后,将纯化的浓缩rdtx和rdtb注射到BALB/c小鼠中,并检测抗体滴度。结果用携带dt和dtb基因的pET-29a(+)对大肠杆菌DH5α和大肠杆菌BL21进行了遗传转化,并在Luria Bertani/卡那霉素培养基上呈阳性生长。发现由1600bp和1000bp组成的dt和dtb序列的开放阅读框分别与白喉杆菌的dt和dtb序列(登录号KX702999.1和KX702993.1)100%相同。高细胞密度的最佳条件是补料分批发酵生产,以280和240Lf/mL表达rdtx和rdtb,溶解氧约为24%和22%,细菌干细胞重量分别为2.41g/L和2.18g/L。结论本研究成功地制备了两株用于生产白喉疫苗的转基因菌株,并达到了理想的生产条件,达到了最高的生产效率。
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来源期刊
CiteScore
3.70
自引率
3.70%
发文量
29
审稿时长
8 weeks
期刊介绍: Clin Exp Vaccine Res, the official English journal of the Korean Vaccine Society, is an international, peer reviewed, and open-access journal. It covers all areas related to vaccines and vaccination. Clin Exp Vaccine Res publishes editorials, review articles, special articles, original articles, case reports, brief communications, and correspondences covering a wide range of clinical and experimental subjects including vaccines and vaccination for human and animals against infectious diseases caused by viruses, bacteria, parasites and tumor. The scope of the journal is to disseminate information that may contribute to elaborate vaccine development and vaccination strategies targeting infectious diseases and tumors in human and animals. Relevant topics range from experimental approaches to (pre)clinical trials for the vaccine research based on, but not limited to, basic laboratory, translational, and (pre)clinical investigations, epidemiology of infectious diseases and progression of all aspects in the health related issues. It is published printed and open accessed online issues (https://ecevr.org) two times per year in 31 January and 31 July. Clin Exp Vaccine Res is linked to many international databases and is made freely available to institutions and individuals worldwide
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