Construction And Expression Of Recombinant Escherichia Coli Heat-labile Enterotoxin And Its Non-toxic Derivatives

Escherichia coli heat-labile enterotoxin (LT) is produced by enterotoxigenic Escherichia coli(ETEC) strains which are the causative agents of traveler's diarrhea. LT is composed of one A subunit with ADP-ribosylating activity, and the homopentameric B subunit containing the monosialoganglioside (GM1) receptor-binding site. LT is a powerful immunogen and a potent mucosal adjuvant as well. However, the toxicity of LT renders it unsuitable for practical human use. Therefore, many attempts have been made to reduce the toxicity of LT while maintaining its powerful immunological activity. Some derivatives have been generated, which have either greatly reduced or undetectable toxicity but retain some adjuvant or immunomodulatory activity.Studies have shown that the fully non-toxic mutant LTK63 and the recombinant LTB(rLTB) are potential immunogens and mucosal adjuvants suitable for use in human vaccines. But there are doubts about the mechanism of action of LT and the universality of rLTB as mucosal adjuvant.In this study, the wild type genes of E44815, elt and eltB, were amplified by PCR technique and the site-directed mutant gene for mLT63 (Ser? Lys) was generated by the technique of over-lap extension PCR. And prokaryotic systems expressing LT, LTB or mLT63 were constructed and analyzed. This research was performed to obtain non-toxic derivatives of LT as antigen candidates of ETEC vaccine or mucosal adjuvant for many other antigens with the aim to supply a basis for the study of the mechanism of action of LT and its application in human vaccines.Methods1. Clone and sequence analysis of elt gene and eltB gene of E44815The big wild type plasmid containing the gene for LT was extracted by a modified method of SDS lysis from the ETEC strain E44815 isolated from human. The wild type genes of E44815, elt and eltB, were amplified from the big wild type plasmid mentioned above by PCR technique and cloned to the pNEB193 vector respectively. The recombinant strains were detected by Ampicillin antibiotic and blue-white screening, and the recombinant plasmids were identified to be containing elt or eltB by the methods of restriction endonuclease digestion, specific PCR and sequencing. The nucleotide sequences of elt and eltB were determined and analyzed by biological software DNAMAN.2. Construction, clone and sequence analysis of the mutant gene for mLT63 By the technique of over-lap extension PCR, the site-directed mutant gene formLT63 (Ser? Lys ) was generated by a TCT(Ser)? AAA(Lys) amino acid triplet code substitution at position 63 in amino acid sequence of LT. The mutant gene, mlt63, was cloned to the pNEB193 vector. The recombinant strain and the recombinant plasmids were detected and identified by the same methods that have been described above. The nucleotide sequence of mlt63 was determined and analyzed by biological software DNAMAN, too.3. Bioinformatics analysis of the sequences of elt and mlt63The bioinformatics software Omiga2.0 and Genbank were employed to analyze the secondary structure and immunological characteristics of the corresponding proteins encoded by the elt gene cloned, the genes for LT reported in Genbank and themlt63 gene.4. Construction of prokaryotic systems expressing LT, LTB and mlt63 respectively, expression and immunological identification of the recombinant proteinsThe elt gene, the mlt63 gene and the eltB gene were purified from recombinant cloning plasmids and inserted into temperature control expression vector pBV220 respectively, and then the recombinant plasmids were transformed into E.coli DH5ct. The proteins encoded by three genes were induced and analyzed by SDS-PAGE. The target proteins were determined by Western blotting with anti-CTB serum of mouse.The coding region of the elt gene, the mlt63 gene and the eltB gene were amplified from recombinant cloning plasmids and inserted into fusion expression vector pMAL-c2X designed to produce maltose-binding protein (MBP) fusions respectively, then the recombinant plasmids containing the fusion of target gene with the malE gene encoding MBP were transformed into E.coli TB1. The proteins encoded by the three fusion genes were induced and analyzed by SDS-PAGE. The target proteins were detected by Western blotting with mouse anti-CTB serum. Results1. Results of cloning and sequence analysis of elt gene and eltB gene of E44815The results were according to expectation. (1) The recombinant plasmid containing the elt gene was digested into two fragments, which were vector fragment (2.7kb), and elt gene fragment (1.3kb). And the 1.3kb elt gene fragment could be amplified by specific PCR. It was demonstrated that recombinant plasmid containing elt gene was constructed successfully. (2) The recombinant plasmid containing the eltB gene was also digested into two fragments, which were the cloning vector fragment (2.7kb) and the eltB gene fragment (0.48kb). And the 0.48kb eltB gene fragment could be amplified by specific PCR. It demonstrated that recombinant plasmid containing eltB gene was constructed successfully. (3) The elt gene and the eltB gene fragments cloned were 1282bp and 467bp respectively. The eltB gene cloned was completely same to the corresponding segment of the elt gene cloned.They are identical to the genes for LT and LTB reported in Genbank respectively. Compared with the sequences of genes for LT reported in Genbank, AB011677 (LTh) and S60731 (LTc), the elt gene cloned shared 99.53~99.61% homology in nucleotide acid sequence. The amino acid analysis indicated that they shared 99.22~99.61% homology in Asubunit and 98.39% homology in B subunit.2. Results of construction, cloning and sequence analysis of the mutant gene for mLT63The results showed that, according to expectation, the recombinant plasmid containing the mlt63 gene was digested into two fragments that were the vector fragment (2.7kb) and the mlt63 gene fragment (1.3kb). And the 1.3kb mlt63 gene fragment could be amplified by specific PCR. It was demonstrated that recombinant plasmid containing the mlt63 gene was constructed successfully. The result of sequence analysis of the mlt63 gene cloned showed that there was a TCT(Ser)? AAA(Lys) amino acid triplet code substitution at position 63 in amino acid sequence of LT and no mutagenesis unexpected.3. Results of bioinformatics analysis of the sequences of elt and mlt63The secondary structure and antigenic analysis of LT indicated that LT have highly conserved feature. In theory, it is feasible and universally applicable for LT as antigen of ETEC vaccine and as mucosal adjuvant. The putative secondary structure and antigenicity of mLT63 differ from LT around the site of mutagenesis.4. Results of construction of prokaryotic systems and expression and immunological identification of the recombinant proteinsThe six recombinant expression systems were constructed successfully, which were E.coli DH5a with recombinant plasmid pBV220-elt, pBV220-eltB or pBV220-mlt63, and E.coli TB1 with recombinant plasmid pMAL-c2X-elt, pMAL-c2X-eltB or pMAL-c2X-mlt63. Three recombinant strains DH5a(pBV-elt), DH5a(pBV-eltB) and DH5a(pBV-mlt63) were induced by 42°C to express the target proteins. The target proteins amounted to only about 3-8% of the total bacterial proteins and existed mainly with inclusion body in E.coli strain which was difficult to purify.The fusion proteins expressed by TBl(pMAL-elt), TBl(pMAL-eltB) and TBl(pMAL-mlt63) could be found by SDS-PAGE. The fusion proteins MBP-LT and MBP-mLT63 amounted to 20% of the total bacterial protein after the recombinant strains induced with IPTG in 25°C for 20h. And MBP-LTB amounted to 9.5% of the total bacterial protein in the same induced condition. The results of Western blot tests showed all the fusion proteins could be specifically recognized by the anti-CTB serum.Conclusion1. The elt gene and the eltB gene of E44815 are cloned successfully and the site-directed mutagenesis of the mlt63 gene cloned is performed correctly.2. The secondary structure and antigenic analysis of LTh and LTc indicate that LT has highly conserved feature. In theory, it is feasible and universally applicable for LT to be antigen of ETEC vaccine and mucosal adjuvant. The putative secondary structure and antigenicity of mLT63 significantly differs from that of LT around the site of mutagenesis, which stands a good chance for reducing or wiping out the enzymatic activity and toxicity of LT.3.The prokaryotic expression systems DH5cc(pBV-elt), DH5a(pBV-eltB) and DH5ct(pBV-mlt63) express a small quantity of target proteins existing mainly with inclusion body, which is unsuitable for purification and application.4. The prokaryotic expression systems TBl(pMAL-lt), TBl(pMAL-ltB) and TBl(pMAL-mlt63) are high efficiency. And the proteins expressed in those recombinant strains are easy for purification. The recombinant strains can be used to study the biologic activity of LT and its value in vaccine preparation. And they also can be taken as candidate strains for preparation the recombinant antigen of vaccine against ETEC and mucosal adjuvant.