Studies On Recombinant Expression Of Novel Conotoxin Gene

Conotoxins are biologically active small peptides isolated from the venom of venomous marine cone snails, which are characterized by the number of their cysteine residues, the arrangment of the disulfide bonds and the distinctive and conserved signal region of the toxin precursor. Conotoxins are utilized as reasearch tools in many fields of neurobiology with high-affinity antagonists for receptor and ion channel subtypes, and being small peptides, they are synthesizable and modifiable, thus increasing their availability and potential utility. As mentioned above they can be used as both pharmacological tools and templates for drug design. Therefore, there is a very important significance as soon as possible to seize the genetic resources of medicinal marine life Conus, when Hainan Conus resources are increasingly endangered in the present situation. There are important theoretical value, broad application prospects and huge potential economic benefits for the study of conotoxin biosynthesis in the field of marine biomedicine.Conotoxins were synthesized through genetic engineering methods, in order to solve the scarcity of natural conotoxin resources, expensive chemical synthesis, separation purification difficulties and other problems. We designed a number of synthetic primers according to novel conotoxin gene, were cloned into the expression vector in four different expression systems, to explore the effect of the conotoxins gene expression in the four kinds of expression systems, to lay a foundation for the structural and functional studies of conotoxins.This study includes the following sections:1. Expression of conotoxin gene GeXIVAWT in Escherichia coliAccording to the conotoxin gene GeXIVAWT, two pairs of primers GeXIVAWT-P1/2and GeXIVAWT-P3/4were designed in accordance with the E.coli codon preference, and annealed to form gene GeXIVAWT-12and GeXIVAWT-34. And gene GeXIVAWT-12including a stop codon, the expression of recombinant conotoxin has a complete C-terminal; gene GeXIVAWT-34with no stop codon, the expression of recombinant conotoxin has a His-tag to facilitate post-purification. They were cloned into digested pET22b(+) vector, and two expression vectors pGeXrVAWT-12and pGeXIVAWT-34were successfully constructed. These two expression vectors were transformed into E. coli BL21(DE3), recombinant conotoxin GeXIVAWT was highly expressed in the form of insoluble inclusion bodies by IPTG induction, after optimization of rGeXIVAWT-34expression level reached61.6mg/L. A large number of recombinant conotoxins GeXIVAWT-12/34were purified by ultrafiltration and affinity column, and they were further purified and identified by mass spectrometry, and E. coli signal peptide failed to effectively resection. Biologically active recombinant conotoxin was obtained by the dilution method of refolding. Activity experiments show that recombinant conotoxin GeXIVAWT-12/34can inhibit the growth of insect cells with a dose effect.2. Expression of conotoxin gene MrVIB in Escherichia coliOne pair of primers MrVIB-P1/2with no codon optimization were designed according to the conotoxin gene MrVIB and annealed to form gene MrVIB-12with no stop codon, and the expression of recombinant conotoxin has a His-tag to facilitate post-purification. Gene MrVIB-12was cloned into digested pET22b(+) vector, and expression vector pMrVIB-12was successfully constructed. This expression vector was transformed into E. coli BL21(DE3), IPTG induction and expression conditions were optimized, the results showed that recombinant conotoxin MrVIB got the secretion expression, a large number of recombinant conotoxin MrVIB was purified by affinity chromatography and identified by mass spectrometry, results show that the signal peptide was cut by endogenous signal peptidase, and then in the periplasmic correctly form three pairs of disulfide bonds. Biological activity experiment showed that the recombinant conotoxin MrVIB could alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.3. Expression of conotoxin gene MrVIB in Pichia pastorisAccording to gene GeXIVAWT, four pairs of primers MrVIB-P5/6, MrVIB-P7/8, MrVIB-P9/10and MrVIB-P11/12were designed and primers MrVIB-P9/10and MrVIB-P11/12were synthesized to be optimized according to the Pichia pastoris preferred codons. Four pairs of primers annealed to form gene MrVIB-56, MrVIB-78, MrVIB-910and MrVIB-1112. Gene MrVIB-56and MrVIB-910with no stop codon, and the expression of recombinant conotoxin has a His-tag to facilitate post-purification; gene MrVIB-78and MrVIB-1112with stop codon, the expression of recombinant conotoxin has a complete C-terminal. These genes were cloned into digested pPICZαA vector, the four yeast expression vectors were successfully constructed for the aA-MrVIB-56,αA-MrVIB-78, aA-MrVIB-910and αA-MrVIB-1112. These expression vectors were linearized and electroporated into Pichia pastoris, high-copy recombinants were screened by antibiotic Zeocin and PCR, and expression was induced by methanol. Induced expression of the conditions were explored, the results show that the medium MGY/MM was better than BMGY/BMMY. The Tricine-SDS-PAGE electrophoresis analysis showed that the recombinant conotoxins MrVIB-56and MrVIB-910were expressed, and recombinant conotoxin MrVIB-910with the optimization of codon expression levels was increased slightly. Therefore, conotoxin gene MrVIB was successfully expressed in Pichia pastoris, but the expression was very low, and expression conditions need to be optimization in the next step.4. Expression of conotoxin gene MrVIB in insect cellTwo primers MrVIB-P13/14was designed according to the conotoxin gene MrVIB, and annealed to form gene MrVIB-1314including a stop codon, the expression of recombinant conotoxin has a complete C-terminal. Gene MrVIB-1314was cloned into digested pFastBacTMTB, and the pFastBac-MrVIB-1314plasmid was successfully constructed. It was transformed into competent E. coli DH10Bac, the gene MrVIB was cloned to Bacmid shuttle vector by the help of helper plasmid, and the plasmid Bacmid-MrVIB-1314was obtained. By liposome-mediated method, the plasmid Bacmid-MrVIB-1314was transfected cells Sf9to express recombinant baculovirus plasmid, and expression products were analyzed by Tricine-SDS-PAGE. The experimental results show that the baculovirus Bacmid-MrVIB-1314was expressed in infected insect cells. Conotoxin gene was expressed in the baculovirus expression system, specific testing and validation need to be further purified and identified by mass spectrometry and biological activity.5. Expression of conotoxin gene MrVIB in mammalian cellTwo pairs of primers MrVIB-17/18and MrVIB-19/20were designed according to the conotoxin gene MrVIB, the gene MrVIB-1718and MrVIB-1920were obtained after annealing. They were respectively cloned to digested pcDNATM4/Max-HisA and pcDNATM3.1/V5-HisA, and we have successfully constructed two expression vectors pcDNA4-MrVIB-1718and pcDNA3.1-MrVIB-1920. The two recombinant plasmids DNA were transfected into CHO cells using liposome transformed cells, resistant cells CHO-1718and CHO-1920were obtained after G418selection, and recombinant conotoxins could be stable expression. The experimental results show that the recombinant conotoxins MrVIB-1718and MrVIB-1920were secreted expression in CHO cells, and were conducive to the expression of post-translational modification, and could get the structure and biological activity of natural conotoxins. Specific testing and validation need to be further purified and identified by mass spectrometry and biological activity.In summary, the conotoxin genes were expressed in E. coli, Pichia pastoris, baculovirus, and mammalian cells four expression system, to explore and establish high-activity, low-cost, securityhigh, large-scale production of recombinant conotoxins exogenous expression system laid the foundation.