The Research Of Prokaryotic Expression And Eukaryotic Expression Of The Porcine Rspo3Gene

Rspo is a newly discovered gene family composed of four gene members (Rspol to Rspo4), which interacts with membrane proteins FZD and LRP5/6to activate Wnt/β-catenin pathway. This interaction is closely associated with cell growth and differentiation, organ development, and tumor metastasis. Rspo gene family is widely distributed in different species with high conservativeness. Among all the Rspo genes, Rspo3is indispensible for individual development of animal, which is indicated by the fact that mice with Rspo3knock-out cannot grow normally to adults. This phenomenon brings obstacles to further study gene functions of Rspo3. Among all the husbandry animals in our country, pig is especially important:studying of Rspo3gene will significantly benefit the new species development and disease control of pigs;In the current study, we used PCR to clone porcine Rspo3gene and successfully expressed this gene in E.Coli BL21strain using pET28a as the vector. To further study the function of Rspo3in eukaryotic cells, eukaryote expressing vector with different promoters was established and screened for gene expression efficiency.1.Using the technology of electronic cloning, the amplification primers for porcine Rspo3was designed. RT-PCR was used successfully to acquire the porcine Rspo3gene with total RNA from pig liver as the template. With confirmation from gene sequencing, the open reading frame of porcine Rspo3has828base pairs and encodes275amino acids, including56acid amino acids and62basic amino acids, and the molecular weight of its expressed protein is31.32kDa with a PI of9.94. The three-dimensional structure of RSPO3was also predicted by us. The protein sizes are: X:44.486Y:45.211Z:47.410(unit:angstrom) and it belongs to globular proteins. The distribution study of Rspo3indicated that it is universally distributed in every organs except spleen and fats, with highest expression level in brain and testicles.2.Porcine Rspo3gene was integrated in a pET28a prokaryotic expression vector, and6X Histine-tag was added for further detection and purification without compromising the folding and immunogenicity of RSPO3. With E.coli BL21as the recipient bacteria, the optimized expression condition was at26℃and0.5mg/ml of IPTG, with induction time of2h. Using SDS-PAGE and Western Blot, we confirmed the expression of fusion protein with the molecular weight of 35.19kDa.3.Rspo3gene was ligated with eukaryotic expression vectors with ten different promoters (P1-P10), however, the NcoI site inside Rspo3served as the major obstacle of normal enzyme-cleavage based ligation. Using SOE-PCR, we conducted samesense mutation of NcoI and confirmed the mutation by gene sequencing:the sequence is unchanged except the NcoI region, and the encoding amino acid sequence is exactly the same to the non-mutant. Mutated Rspo3gene was ligated with P1-P10after double enzyme cleavage by NcoI and NheI from T vector. With the confirmation of PCR, enzyme cleavage, and gene sequencing, successful eukaryotic expression vectors carrying Rspo3gene and10different promoters were established.4.The transfection experimental results in PK15cells demonstrated that P5plasmid has the highest expression efficiency for GFP expression:458.9%increase was observed compared with control group using peGFP-N1plasmid, and GFP fluorescence was observable even on day14post-transfection.P6plasmid has the second highest efficiency for GFP expression. With Q-PCR detection, the most suitable promoter combination for PK15cells were CMV(Human)+HTLV RU5’(Viral)+CMV(Human) carried by P5plasmid.5.Similar promoter screening was also conducted in chicken fibroblast DF1, canine kidney cell MDCK, and human ovarian cancer cell line Hela. By fluorescence microscopy detection, fluorscence absorbance test and expression lasting time measurement, we confirmed that P6plasmid has the highest expression efficiency in DF1and MDCK cells, with the promoter combinations of CMV(Human)+HTLV RU5’(Viral)+Sv40(Viral). At the same time, P5plasmid has the highest expression efficiency in Hela cells, with the promoter combinations of CMV(Human)+HTLV RU5’(Viral)+CMV(Human).In summary, we successfully cloned the porcine Rspo3gene and evaluated its distribution in different organs. The protein was successfully acquired in E.Coli BL21strain. Eukaryotic expressing vectors with different promoter-combination were established and screened in4different cell lines for expression efficiency. These studies provided the foundation for further research of Rspo3gene functions and clarification of its applicability in husbandry animals.