Preparation Of Recombinant Human Serum Albumin And Establishment Of Double-Antibody Sandwich ELISA Method

Human serum albumin usually exists as a soluble protein monomer synthesized by liver cells. It accounts for60%of the total protein in plasma, and has many important physiological functions, like maintaining blood colloid osmotic pressure, transportation, nutrition, and used to treat massive haemorrhage, acute blood volume decreases and hypoproteinemia clinically. In addition, it can also be used as the auxiliary material in pharmaceutical industry, such as the medium of eukaryotic cell cultures, the protein protectants and excipients. Its usage becomes the first in the international market.The current clinical used HSA was mainly separated from human blood, but with the shortage of blood resources and the existing pollutions, looking for rich and safe HSA becomes more urgent. More and more people tend to express rHSA for further use, especially in clinically quantitative diagnosis. Therefore, it has great significance in the establishment of fast, practical, specific and sensitive detection method of human serum albumin. So, we cloned HSA gene and express it in E. coli. With the purified HSA, we prepared polyclonal antibody, and established a double antibody sandwich ELISA method to test HSA, and lay the foundation of HSA detection of genetically engineered, human serum and plasma existed. The results are as follows:1. According to the published HSA nucleotide sequence in GenBank（NM₀00477.5）, we cloned the1,755bp HSA gene from human embryo lung diploid cells by RT-PCR and then cloned it into pGEX-4T-1vector. The results showed that, the nucleotide sequence homology between sequencing pGEX-4T-1-rHSA and human serum albumin cDNA in GenBank was100%. Then the HSA cDNA sequence was successfully cloned, and the expressed HSA can react with specific antibody.2. The recombinant HSA was purified with GST FF affinity chromatography agarose gel after denaturation and renaturation, and then immunized rabbits as immunogen to get HSA polyclonal antibody. After gel chromatography purification, the antibody was identified by indirect ELISA and Western blotting. The results showed that the recombinant HSA mainly exists as inclusion body with good reactivity and immunogenicity, and the molecular weight was about93kD. The antiserum titer could reach1:100,000, and it could react with rHSA and the HSA from blood with good biological activities.3. With the help of mouse monoclonal antibody and rabbit polyclonal antibody, we established the HSA double antibody sandwich ELISA detection method. The linear detection range was0.4μg/mL purified polyclonal antibody, and10mg/mL bovine albumin as block buffer, and1:20,000HRP-conjugated mouse anti-human serum albumin antibody. The sample and HRP-conjugated antibody reaction time were both60min, and the chromogenic time was15min. This method had good specificity and repeatability, which could lay the foundation for the establishment of a quick and easy HSA detection method.In conclusion, this study successfully constructed the HSA prokaryotic expression vector, prepared HSA rabbit polyclonal antibody, and established a double antibody sandwich ELISA detection method for future HSA diagnostic tests.