[Prokaryotic expression of F10 and preparation of its polyclonal antibody].

AIM: To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it. METHODS: The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA. RESULTS: Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000. CONCLUSION: F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.

[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

A novel mutation of estrogen receptor gene detected in girls with precocious puberty.

Female precocious puberty is caused by premature activation of the hypothalamic-pituitary-gonadal axis, exposure to exogenous sex steroid hormones, and the presence of endogenous sex steroids caused by various factors. Estrogen is the final key factor to start onset of puberty. However,in some cases of precocious puberty in girls estrogen elevation could not be detected. The raised sensitivity of estrogen receptor, which may caused by ESR1 mutation or polymorphism, has been frequently mentioned for interpreting the etiology of sporadic low estrogen type cases. But no case evidence has been found in clinical practice. For the purpose of screening possible mutations in estrogen receptor gene, leukocyte genomic DNA were collected from 16 girls with precocious puberty of sporadic low estrogen,and exons of ESR1 were amplified and analysized using PCR-SSCP/silver staining method. A single strand conformation change in exon 8 was found in one of the patients (No. 14). The suspected fragment were cloned to a T vector and sequenced for analysis. Sequencing of these clones revealed that this conformation change is caused by a C to T transition. This mutation results in the replacement of arginine by cystine at position 548 of ESR1 protein. The mutation created an extra Btsl digest site and made it can be readily identified by PCR-PFLP method. Further detection using this method, and sequencing of cloned exon8 colonies from patients proved that the patient No. 14 is Arg548/Cys548 heterozagous in genotype. This mutation increased hydrophobility of the area dramatically. The position and the conservative of this residue in vertebrates suggested Arg548 may play an important role in ESR1 function. For study the role of this mutation in the onset of precocious puberty, a firefly luciferase reporter plasmid pGL3-promoter-ERE was constructed,and a pCR3. 1-hermut pisimid expressing Cys548 ER was constructed based on wild type pCR3. 1her. Co-transfection of reporter and pCR3. 1 -hermut in CMF-7 cell strain proved that Cys548 mutant can significantly increase the transcription activity over the Arg548 wild type.

[Association of the novel hydatidiform mole-related gene F10 with the invasiveness of trophoblastic tumor].

OBJECTIVE: To study the expressions of the novel gene F10 associated with hydatidiform mole in different trophoblastic tumors and explore the relation of F10 expression with the invasiveness of malignant trophoblastic tumor. METHODS: In situ hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole, 6 cases of invasive mole, and 8 cases of choriocarcinoma. RESULTS: F10 mRNA was positive in all cases of hydatidiform mole, invasive mole, and choriocarcinoma, and the expression intensity significantly increased in the order of hydatidiform mole, invasive mole and choriocarcinoma (P<0.001). CONCLUSION: The expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor, with possible involvement in the invasion or malignant changes of trophoblastic cells.