Expression And ELISA Analysis Of The Recombinant Rats G-protein Coupled Receptors Kinase 5

GRK5 is a G protein-coupled receptor kinase(GRKs),which is a member of the kinase family,phosphorylate the G protein-coupled receptors(GPCRs). GPCRs is the largest transmembrane receptor in organisms.GRK5 mediat the receptor desensitization,regulating the signals transduction which play an important role in the differentiation and the process of disease.The studies have shown that GRK5 has a very important role in differentiation of thyroid cancer, hypertension,heart failure,Alzheimer disease,Parkinson's disease,memory function and drug addiction.However,the specific role of GRK5 in these diseases is not very clear.The previous studies of GRK5's specific function were just proved by animal models.Advantages of animal models:the abnormal disease can be difficult to see in clinical but can been returned in animal models, you can strictly control the experimental conditions to enhance the comparability of the experimental materials,contribute to a more comprehensive understanding of the nature of the disease;The shortcomings are: time-consuming,hard sledding,a longer cycle.Gene cloning technology enables Eukaryotic gene can be expressed in Prokaryotic expression system.In this system,purify protein is also relatively easy,which will be a convenience to research the protein activity.Prokaryotic expression system is easy to operate, fast and takes shorter time to express a large amount of products etc.We can transfere and express GRK5 in Prokaryotic expression systems by gene cloning technology.In Prokaryotic expression systems,the protein can be purified more conveniently,which is important in the following research.Our experiment is to express the GRK5 of SD rats in E.coli.1.GRK5 gene amplification,cloning and sequence analysisAccording to the GeneBank database published the SD(Sprague-dawley) rats of the GRK5 gene sequences,we designed the specific primer of the GRK5 gene,then extracted the total RNA from SD rat,amplified GRK5 gene by RT-PCR,digested the product and cloned into vector pBluescriptâ…¡SK(+),sent to the company to sequencing,blast the nucleotide sequence of GRK5 in GeneBank database,as a result,GRK5 gene was been cloning successed.2.GRK5-GST recombinant protein expressed and optimized in E.coli Rosetta-gami(DE3)GRK5 gene recombinant to the pGEX-4T-1 vector was transformed into E.coli Rosetta-gami(DE3) induced when OD₆₀₀ is up to 0.6,IPTG's final concentration of 0.3mM,37℃,4h later,SDS-PAGE analysis,GRK5-GST recombinant protein express successfully.pGEX-4T-1 / GRK5 recombinant was transformed into E.coli Rosetta-gami (DE3),at the IPTG concentration of 0.3mM,0.5 mM,0.7 mM,0.9 mM,induced by a temperature of 25℃,28℃,30℃,37℃,respectively 3h,4h,5h,6h later, collect the bacteria,after SDS-PAGE and ELISA analysis,there is no change in expression and the expression level was very low,which are probably caused by a variety of reasons,such as:rare codons,eukaryotic genes express in Prokaryotic system.3.ELISA used to detect the expression level of GRK5Immunodiagnostics ELISA is a new technology,which is sensitive,specific, simple,fast,stable and easy to operate,such as automation.This experiment,the samples containing GRK5 with the coating liquid according to the ratio of 1:2 dilution;the GRK5 polyclonal antibody concentration by the dilution ratio 1:500, 1:600,1:700,1:800,1:900,1:1000 to optimization the suit concentration of the first antibody;HRP labeled goat anti-rabbit secondary antibody was according to the instructions recommend the best concentration ratio of 1:500 dilution.At the same time,We set up blank and negative controls,anti-double-detection of the expression level of GRK5,measured absorbance of the microplate at 450nm, then calculating the S / N value.ELISA results showed that GRK5 sucessifully express in E.coli Rosetta-gami(DE3),which can be a basis for further researches.