| Background:Cervical cancer is a common and frequently-occurring disease in developing country, and already the second of malignant tumor, second only to breast cancer. According to the WHO estimination, the number of new cervical cancer cases in China accouted for about 28% of total cases in the world each year. Cervical cancer has become a major threat to women’s health and now rise to a social and public health problem. But the prognosis and curative effect is good through the early detection and timely treatment. The literature reports the 5-year survival rate of early cervical cancer can amount to 92%. So it’s very important for cervical cancer to early detection, early diagnosis and treatment. At present the diagnosis methods of cervical cancer including traditional cervical smear cytology, colposcopy examination and histological examination. These methods compared with the previous improve the early diagnosis of cervical cancer, but these methods still have their own defects,such as pap smear test is less sensitive, colposcopy examination is difficult to check the small lesions and biopsy is invasive examination, it causes poor patient compliance.In recent years, tumor markers such as carbohydrate antigen 19-9, squamous cell carcinoma tumor-associated antigen, carbohydrate antigen 125, carcinoembryonic antigen has been auxiliary detection of cervical cancer, but this tumor markers is less ensitivity and specificity for cervical cancer. So to find high sensitivity and specificity of new tumor markers for cervical cancer is particularly important. The phage display technology (PDT) was invented by Smith, it provide the conditions for screen tumor markers of cervical cancer. In this study,we use phage display technology to binding with cervical cancer, screening the serum peptides with high affinity.Objective:Using Ph.D-12TM phage random peptide library screening of cervical cancer patients serum, and screening the phage clones of specifically binding with serum of cervical cancer patients. Use ELISA test to verify the binding situations of specificity phages by expanding sample of serum and in other tumor serums, screening the best phage clones of specifically binding with patients serum of cervical cancer, DNA sequencing of phage clones were screened, and obtain the short peptide sequence according to inserted into the short peptide. Expected for the diagnosis of cervical cancer to provide new tumor markers and to lay a foundation for further study of tumor markers associated with cervical cancer.Methods:1. We collected 6 cases flesh serum of normal people and early cervical patients, and coat the serum in the microporous separately. The phage display peptide library adsorbed with normal serum firstly, then the uncombined phage were adsorbed with the serum of cervical cancer. Elute the phage of specific binding with cervical cancer serum, and use amplification to cultivate phage. Repeat three rounds of screening, expanded specific phages binding of serum of patients of colorectal cancer, finally obtain the specificity of phages.2. Pick up several of individual plaques from flat and use ELISA method to detect the adhesion strength of selected phages with the serum of early cervical cancer. Selection differential have obvious difference and the stability of cyanophage isolates, get individual phage clones with high specificity.3. Choose several of high affinity of phages and repeat ELISA with expanding the sample of serum, finally obtain higher specificity of phage clones.4. Extraction and purification of the phage clones were sequenced, finally according to the insertion loci and genetic code table, obtain the short peptide sequence.Results:, The eluted phages were enriched nearly to 100 times through three rounds of screening, Pick a single phage and enlarge sample size to identify repeatedly, finally obtain the NO.7phage clone of specific binding with cervical cancer serum. The results of ELISA about NO.7phage clones of specific binding with 6 cases serum of normal people and early cervical patients is that the OD values of normal group was 0.056±0.010, the cervical cancer group was 0.151±0.040, P<0.05, the statistical analysis was statistically significant;the results of ELISA about NO.7phage clones of specific binding with 50 cases serum of normal people and early cervical patients is that the OD values of normal group was 0.070 ± 0.023, the colorectal cancer group was 0.157 ± 0.040, P<0.05, the statistical analysis was statistically significant; Use ELISA test to verify the binding situations of the NO.7 phage clones in other tumor serums, the group of cervical cancer was 0.174 ± 0.018, the ovarian cancer was 0.052±0.007, intraepithelial neoplasia group was 0.076±0.010, normal group was 0.052±0.006, the statistical analysis was statistically significant between cervical cancer group and the other groups. The area under the ROC curve of the NO.7 phage clones was 0.956, P< 0.0001,the statistical analysis was statistically significant,95% confidence interval of ROC curve area was [0.896,0.987];use Medcalc software to analyze, the specificity of NO.7 phage clones was 94%and sensitivity was 84%; the sequence of inserted gene was 5’-ATG CAC CGG ACG CGT CAG CGA ACC ATT CGT AGC CGA-3’,the amino acid sequence:SATNGSLTRPVH. Thought analysis of BLAST protein database, the result of DNA sequencing of NO.7 phage clones compared with humans, rats and homologous protein is that without the same sequence and the homologous protein stuctures are consistent with this short peptide.Conclusion:Specific and high-affinity peptides for cervical cancer may be screened successfully, this peptides can be laid the basis for early diagnosis, prognosis and recurrence detection of cervical cancer and further study of tumor marker of cervical cancer.It’s also contribute to study the mechanism of cervical cancer. |
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