The Cloning Expression And Serologic Study Of Mammaglobin And Mammastatin In Breast Cancer

Objective Mammaglobin(or a human mammaglobin,hMAM)has been found in recent years to be a tissue specific protein that is related with breast cell growth,and mammastatin is another tissue specific protein as breast growth inhibitor.The two proteins have been proved that they could been detected in serum.So far,there are less serologic study about the proteins and it's diagnostic significancy for breast cancer keep unclear.Our purpose is to establish a novel sera ELISA by cloning expression and antibody preparation to detect the two proteins in breast cancer patients and healthy women,and analyze the sera expression of mammaglobin and mammastatin,and it's diagnostic implications for breast cancer.Methods The amino acid sequence of mammaglobin and mammastatin were analyzed by BioSun to fix on the epitope sequence,the total gene of mammaglobin and the epitope gene were composed by PCR.The expression vectors were constructed and transformed into E.coli.HB101 for expression.The monoclonal antibodys of mammaglobin were prepared by hybridoma technique.The rabbit was immunized with the purified recombinant protein to prepare aitiserum and was used to detect the serum of normal people and breast cancer patients by ELISA.Results:1.The total gene of mammaglobin was composed by PCR.We successfully constructed two mammaglobin expression vectors which linked with different vectors:pBVIL1/mammaglobin and pGEX-4T-2/mammaglobin,All showed high expression.Two hybridoma cell lines were obtained after the mice were immunized by pGEX-4T-2/mammaglobin,named 1E12 and 4B11.The microtiter plates was coated by 1E12 and biotin was labeled by 4B11.The ELISA was successfully established.We detected the serum of 127 breast cancer patients and 133 healthy women,the result showed the sera expression level of mammaglobin in breast cancer group(average OD value 0.645±0.223)is significantly higher than that in normal control group(average OD value 0.255±0.109),P=0.001.We did't found the statistical differency between the sera expression level of mammaglobin and age,tumor size,clinical stage,axilla lymphnode metastasis,ER in breast cancer patients(P＞0.05). 2.The epitope gene of mammastatin was composed by PCR.We successfully constructed two mammastatin expression vectors which linked with different vectors:pBVIL1—Mammastatin(12a)and pBVIL6—Mammastatin(4a),All showed high expression in E.coli.HB101.The rabbit were immunized with the purified recombinant proteins of pBVIL1—Mammastatin(12a)and pBVIL6—Mammastatin(4a) to prepare aitiserum,the former coated microtiter plates and the later labeled HRP,a steady ELISA was established.We detected the serum of 95 breast cancer patients and 95 healthy women,the result showed the sera expression level of mammastatin in breast cancer group(average OD value 0.135)is significantly lower than that in normal control group(average OD value 0.261),P＜0.01.We did't found the statistical differency between the sera expression level of mammastatin and age,tumor size,clinical stage, axilla lymphnode metastasis,ER in breast cancer patients(P＞0.05)Conclusion Our study confirmed that the proteins of mammaglobin and mammastatin can been detected in the serum by a novel,noninvasive ELISA.We found that the expression of the two biomarkers in the serum of breast cancer patients and healthy women exist significant differency,however,has no relationship with the prognosis of brast cancer.The primary results indicate that the two biomarkers may be useful for early detection of breast cancer.