Isolated FSH Deficiency: In Vitro Validation Experiments Of Wild-type And Mutant P.Arg97X FSH? Subunit

Objective:To lay the foundation for studying the relationship between the mutation and immunological and bioactivity of FSH, and to provide the basis for revealing pictures of FSH physiological functions and spatial structure, we have created the cell model which can express mutant Arg97 XFSH by using the technology of gene amplification, vector construction and cell transfection.Methods: 1. Primers designation: According to NCBI, a pair of primers of FSH? were designed. 2. Gene amplification: RNA was extracted from human's uterine fibroid tissue. As a template, DNA was obtained and purified to gain FSH?gene fragment. 3. Vector construction: FSH? fragment and pc DNA3.1(+) vector were double digested by Hind III, Eco RI, respectively. After that, FSH? fragment was combined with the pc DNA3.1(+) corresponding to the restriction sites by using T4 ligase to obtain pc DNA3.1(+)-FSH ?. The pc DNA3.1(+)-FSH?(WT) and pc DNA3.1(+)-FSH?(MU) vectors had been obtained already. 4.Cell cotransfection,: The three plasmid groups: pc DNA3.1(+)-FSH ? and pc DNA3.1(+)-FSH?(WT), pc DNA3.1(+)-FSH? and pc DNA3.1(+)-FSH?(MU),and pc DNA3.1(+) empty(CK)vector were transiently transfected into Chinese hamsterovary cells. 5. PCR, western-blot, chemiluminescence method: After 48 hours of incubation, cell lysates were extracted for PCR and western-blot detection, cell culture supernatants were collected for FSH ELISA. 6.c AMP concentration: The COV434 cells were cultivated with above cell culture supernatants and complete medium, respectively. After 6 hours of cultivation, cell lysates were extracted for c AMP concentration to test biological activity of FSH.Results: 1. PCR results: The wild-type m RNA and FSH? m RNA were detected in WT group, suggesting that the wild-type FSH?and FSH? were successfully transcribed in CHO; The mutant m RNA and FSH? m RNA were detected in MU group, suggesting that the mutant FSH?and FSH? were successfully transcribed in CHO; Neither FSH? m RNA nor FSH?m RNA was detected in CK group, suggesting that there was no transcription occured. 2. western blot results: The protein of FSH was detected in WT and MU group, suggesting that wild-type and mutant FSH can be successfully translated in CHO; No protein was detected in CK group. 3. Chemiluminescence results: The wild-type FSH concentration was significantly higher than the other groups. 4. c AMP concentration: The c AMP concentration of WT group was significantly higher than the other groups.Conclusions:1. pc DNA3.1(+)-FSH?vector was successfully constructed. Wild-type FSH?gene, mutant FSH?gene and FSH?gene can be successfully transcripted and expressed in CHO. 2. Wild-type FSH subunit can be secreted, suggesting that FSH can be secreted extracellularly and play immunological and biological function only in the situation that ?and ?conbine together. 3. The p.Arg97 X FSH? subunit mutant do not have detrimental impact on the transcription and expression of FSH? subunit. While, immunological activity and bioactivity of mutant FSH can not be detected in vitro, suggesting that the presence of the mutation could destroy the formation of ?-?heterodimer and affect the immunological activity and bioactivity of FSH.