Method of determining klotho protein in human blood

[0023] 1. Construction of human Klotho protein expression vector

[0024] 1. Cloning of the C-terminal gene of human Klotho secreted protein

[0025] A pair of primers were designed based on Human Klotho cDNA sequence:

[0026] Sense primer:

[0027] 5'>GTC CCC GGA TCC GTG TCC ATT GCC CTA A<3′

[0028] Anti-sense primer:

[0029] 5′>ACT AGC CTC GAG ATC TCC AGA GCC GAA A<3′

[0030] A 1.5 kb fragment cDNA was amplified from human kidney tissue RNA using primers.

[0031] 2. Construction of expression vector

[0032] The above-mentioned amplified DNA fragment was cloned into pMD18-T vector, and it was verified by sequencing that it belonged to the C-terminal form of Klotho secreted protein (KLSC for short), and then digested with double enzymes (BamHI and HindIII) from the pMD18-T-KLSC plasmid, The 1.2kb fragment was excised and cloned into pET-30a and pET28a vectors to form the KLSC expression vector. Then these vectors were transformed into E. coli BL21 strain to p...