Construction, Prokaryotic Expression, Bioactive Evaluation Of Fusion Gene Cecrop-m2A10 And Construction Of Its Transgenic System

Background:Mosquitoes transmit numerous human diseases,such as malaria, epidemic encephalitis B,fiariasis,dengue fever,etc,severely harm to human health. An increasing trend on the rate of dengue fever has appeared year by year,occasionally,it might burst out and spread in some regions of the world.Malaria is one of the attacks that result in the highest infection ratio and mortality,taking a heavy toll on the human population in many parts of the world by infecting more than 300 million and killing more than 200 million people each year.The major reasons for this tragic situation are the unavailability of effective vaccines for malaria and the development of insecticide and drug resistance by the vectors and pathogens,respectively.Therefore,there is an urgent need to explore every possible avenue for developing novel control strategies against these mosquito-borne menacing diseases,and at the same time,drug resistance of malaria means that controlling of mosquito becomes the most effective and pragmatic way to ralieve the burden caused by malaria. Plasmodium,the causative agent of malaria,has to complete a complex developmental program in its mosquito host for transmission to occur.Malaria sporozoites exhibit infectivity for mosquito salivary glands,human body and other vertebrate host tissue.The surface membrane of sporozoites of Plasmodium berghei—circumsporozoite protein(CSP) which covers the entire surface of mature,salivary gland sporozoites is a protective antigen of sporozoites.Meanwhile,it may play a role in the adhesion and invasion into the target cell.Mouse monoclonal antibodies against CSP of Plasmodium berghei—2A10 can affect mosquito salivary gland and human liver cell invasion by sporozoites in vitro assays and prevent malaria transmission.2A10scFv is a single chain antibody against CSP of Plasmodium falciparum.CHEN had successfully transformed a 2A10scFv gene into An.stephensi. Transgenic mosquitoes carrying 2A10scFv transgenes exhibited resistance to mosquito salivary gland invasion,the RT-PCR and Northern blot showed that the transgene transcribed well,but Western blot did not detect the protein(Unpublished data)which may be at very low levels in the transgenic mosquitoes.In recent years,the prospect of using transgenic mosquitoes is rapidly gaining strength,owing to the development of molecular biology,immunology and genetics. The central event in transgenic mosquitoes is the heterologous expression of transgenes,expression levels of heterologous genes are crucial to their function. There are many factors may affect the expression level,such as the promoter,coding sequence,the copy number,insertion place and the nature of the expressed protein itself.The most frequently used codons among various species are different. Differences in codon usage can seriously hamper the expression of the heterologous transgenes.This is due,in part,to differences in codon usage,which can significantly result in the potential depletion of tRNAs encoding the same amino acid,decrease translation efficiency and thereby lower protein production in a heterologous system. Suggesting that codon optimization is a valuable strategy for improving the heterologous expression of native sequences,it has been widely and successfully used not only in P.pastoris,but also in other expression system such as E.coli BL21(DE3), Aspergillus niger,and mammalian cells.Various experimental procedures about PCR-based codon optimization have been developed,from the modification of a few codons about 5'codon adaptation to the extensive rewriting of up 1000 bp of DNA by overlap PCR.Likewise,the compatibility of codon usage between the 2A10scFv gene sequence and that of the expression host-mosquito are indeed respectively distinct. Then,if the DNA sequence encoding 2A10scFv gene was designed and synthesized based on the codon bias of mosqiuto,the transgenic expression may be at a higher level and the anti-pathogen effect of transgenic mosqiuto may be stronger.Antimicrobial peptides(AMPs) are small molecular weight proteins with broad spectrum antimicrobial activity against a broad range of micro-organisms,including gram-negative and gram-positive bacteria,fungi,and viruses.They were found in plants,insects,bacteria and hunman.An expansive list of unique AMPs has been discovered in our environment,with well over 800 peptides known.Once in a target microbial membrane,the peptide kills target cells through diverse mechanisms. Cathelicidins and defensins are major groups of epidermal AMPs.Cecropins are one family of these peptides which were first isolated from the haemolymph of the giant silk moth,Hyalophora eecropia and may be idespread throughout the animal kingdom.The principle insect cecropins(A,B and D) are 35 to 37 residues,devoid of cysteine,with a strongly basic N-terminal linked to a neutral C-terminal by a flexible glycine-proline link.The overall structure deduced by NMR for cecropin A is two near perfect amphipathic segments joined by a Gly-Pro hinge.Antibacterial activity has been reported for cecropins A,B and P gainst Gram-positive and Gram-negative organisms.There are some problems in direct expression of AMPs for the following reasons:①In the case of direct expression of AMPs,undesirable degradation of such small sized mRNA and peptide was expected.②The direct expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases.To overcome these potential problems,a common strategy to achieve an efficient expression of a protein or antimicrobial peptide is to express the molecule in a microbial system,either de novo or as a fusion partner with another protein.The gene fusion was successfully translated in a bacterial expression system.The fusion protein was non-toxic to the host bacteria. Our researches suggest that the strategy investigated in this study is useful to produce a large amount of AMPs and it might be applicable to other cationic small peptides especially to those which are difficult to synthesize chemically and have no available affinity purification methods.Consequently,in order to increase the anti-pathogen effect and stability of 2A 10scFv gene,we designed to linked 2A 10scFv gene with the coding sequence from A.gambiae cecropin A and constructed a fusion gene Cecrop-m2A10.Objective:1.To modify coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum and construct fusion gene Cecrop-m2A10.2.To express Cecrop-m2A10 in E.coli BL21(DE3) and analyze the bioactivity of the expression products.3.To construct pBac-AsVg-Cecrop-m2A10 plasmid which contains a full regulation sequence,3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter and Cecrop-m2A10 gene.Methods:1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons and then linked with the coding sequence from A.gambiae Cecropin A.The fusion gene Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was constructed.2.Completed Cecrop-m2A10 gene was cloned from pBSK(+)-Cecrop-m2A10 plasmid by PCR,Cecrop-m2A10 gene fragment was cloned into pET32a(+) vector to construct recombinant plasmid pET32a(+)-Cecrop-m2A10 which was identifed by sequence analysis.3.The plasmid pET32a(+)-Cecrop-m2A10 was transformed into E.coli BL21(DE3) to express the recombinant proteins after the induction of IPTG.4.Optimize expression conditions and purify expression products.5.Identify the recombinant proteins by Western blotting.6.The bioactivity of the expression products was assayed with Argarose-Diffusion-Method.7.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was constructed by techniques of molecular cloning such as digestion and ligation methods with the basic plasmid:pBSK(+)-Cecrop-m2A 10 and pSLfall80fa-AsVg-CFP-3'VTR.Results:1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons.Totally,170 nucleotides in six kinds of amino acids were replaced and then linked with the coding sequence from A.gambiae Cecropin A.Subsequently,the fusion gene,Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was successfully constructed. 2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and its open frame was corrected by sequence analysis.After IPTG inducting,recombinant protien was expressed in inclusion body form in E.coli BL21(DE3).After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.Western blotting showed the recombinant protien could be specifically recognized by anti-6-His antibody.3.The argarose-Diffusion-Method showed bacteriostasis inhibition to the growth of E.coli DH5α.4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.Conclusion:1.The fusion gene Cecrop-m2A10 was successfully constructed.2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and expressed in inclusion body form in E.coli BL21(DE3). After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.3.The preliminary evaluation showed bacteriostasis inhibition to the growth of E.coli DH5α.4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.