Preparation Of The Recombinant Aminotransferase Standard Materials And Human Cytomegalovirus (HCMV) IgG Reference Materials Panel

Objective:To prepare the recombinant aminotransferase standard material for the quality control of clinical laboratory and aminotransferase detection kits. The protein of alanine aminotransferase and aspartate aminotransferase is obtained by the technical of gene recombinant. The standard materials is produced by add the protein of alanine aminotransferase and aspartate aminotransferase to the serum. To establish the reference materials panel for human cytomegalovirus IgG antibody detection kits.The reference materials can be used for the quality control of clinical laboratory and human cytomegalovirus IgG antibody detection kits.Methods:The first part:The Preparation of the Recombinant Aminotransferase Standard Materials.1. The acquired of the gene sequence.The gene of alanine aminotransferase and aspartate aminotransferase was searched in the website of NCBI. The protein was wanted to be expressed by Escherichia coli,in order to obtain much of the aiming protein, the codon of the ALT and AST was optimized according to the E. coli codon usage analyzer. Then, the gene sequence which be optimized was be synthetized in the biotech company.2. The construction of recombinant strain for protein expression. The target gene was digested by Restriction endonuclease BamH Ⅰ/EcoR Ⅰ and insert the gene into vector PRSF-Duet which was also digested by Restriction endonuclease BamH I/EcoR I to construct PRSF-Duet-ALT and PRSF-Duet-AST. Then, the expression vector was transformed into Escherichia coli.3. The protein expression and purification. The E. coli was induced expression protein by IPTG. The protein was purified from cells lysate by Affinity chromatography and gel-filtration chromatography. The alanine aminotransferase and aspartate aminotransferase was obtained through the protein purification, then, the activity of the alanine aminotransferase and aspartate aminotransferase was detected.4. The treatment of the serum. The matrix of the aminotransferase standard material was normal serum. In order to insurance the biological safety, the blood screening test was carried out. The Fibrinogen in the serum was removed.5. The preparation of the standard material. Add the protein of alanine aminotransferase and aspartate aminotransferase to the serum, mixed well, and distribute the mixture to the freezing tube.6. The dilution of the standard material. The standard material was diluted by serum and physiological saline, then the sample was tested.7. The evaluation of the aminotransferase standard material. A series of studies was carried out to evaluate the character of the standard material, The studies included the evaluation of homogeneity and stability8. The value assignment and uncertainty budgets of the standard material. The aminotransferase standard material was certified for the catalytic activity concentration of ALT and AST. Three different laboratories were participated to assign the value of the standard material using IFCC reference procedure. Then, the uncertainty of the standard material was be estimated.The second part:The establishment of human cytomegalovirus IgG reference materials.9. The reference materials were produced by normal serum. The Fibrinogen in the serum was removed. The10different sources of positive serum and5different sources of negative serum were select by kits.10. The confirmation and re-checked of the serum. The technique of indirectimmunofluorescence and western blot were used to confirm the serums. The serums were re-checked by different human cytomegalovirus IgG detect kits.11、The verification of the reference materials panel. Six different human cytomegalovirus IgG detection kits which produced at home and abroad were used to verify the reference materials panel. The verification of the reference materials panel included three aspect named accuracy、specificity and sensitivity.12. The research of the reference materials panel for stability. The reference materials panel was treated in different conditions.then, the treated panel was detected by human cytomegalovirus IgG detect kit.Results:The recombinant strain for protein expression was constructed successfully.The alanine aminotransferase and aspartate aminotransferase was obtained by the protein expression and purification. The catalysis activity of the alanine aminotransferase was80000U/L and the catalysis activity of the aspartate aminotransferase was136000U/L. The aminotransferase standard material was made with the protein of alanine aminotransferase and aspartate aminotransferase. The result of the standard material evaluation display that the standard material had good homogeneity and stability. Through the study of value assignment and uncertainty budgets the catalysis activity of ALT in the standard was1020.5U/L, the uncertainty was194.40U/L, the catalysis activity of AST in the standard was792.9U/L, the uncertainty was78.37U/L.As respected the result of the indirectimmunofluorescence, western blot and the re-checked of the serum was consistent with the result of serum screening. The result of accuracy, specificity and sensitivity verified by detection kits of six different companies matched the requirements. The research of stability indicated these panels were stable enough.Conclusions:The aminotransferase standard material and the human cytomegalovirus IgG reference material was established successfully, they can be suitable for the quality control of clinical laboratory and in vitro diagnostic reagents.