Preparation And Preliminary Application Of The Monoclonal Antibody Agaist NPM1Protein Up-regulated Expression Of The Colorectal Cancer

Colorectal cancer （CRC） is a common malignant tumor of digestive tractwhich seriously endangers people’s health. In America, the number of people livingwith the colorectal cancer ranks third, next to those of the skin cancer and lung cancer.However, in recent China, the morbidity of the colorectal cancer positions fourthamong the common tumor, behind the lung cancer, gastric cancer and liver cancer. Inrecent years, an increasing age at onset of the colorectal cancer has variouslyadvanced, with its incidence and mortality showing an upward trend. Every year, upto940,000people suffer from new cases of the colorectal cancer and almost50,000people die of the illness. With improvement of the medical care, the5-year survivalrate after the colorectal cancer surgery has been greatly improved during the20thcentury but hovers between50%~70%for much of this decade. For thecolorectal cancer patients with no lymph node metastasis, the5-year survival rate afterthe surgery is90%, while for those with lymph node metastasis, the survival rate afterthe surgery is only65%. Therefore, early detection, early diagnosis and earlytreatment are crucial to improve the curative effect and prognosis. Besides, it is ofgreat significance to augment the quality of life and prolong patients’life.To seek early effective diagnostic methods of the malignant tumor is always oneof the aims of medical researches. At present, the molecular marker has aroused wide concern of the academic world because of its important role in diagnosing, curing andpreventing the malignant tumor. The tumor marker(TM) is a kind of active substancegenerated by the cancer cell, most of which are non-specific associated antigen.To detect the tumor marker, which is one of the main auxiliary examinations ondiagnosing the colorectal cancer, is widely applied. With simple and rapid operationsas well as small trauma, it is easy to be accepted. Many scholars have done a plenty ofresearches on the value of diagnosing the colon cancer, and lots of documentliteratures show that the tumor markers, such as carcino-embryonic antigen, CA72-4、CA19-9、CA125、 CA242have high expressions in the colon cancer. The single andjoint monitoring possesses certain clinical application value in screening thecolorectal cancer. However till date, no single tumor marker with high sensitivity andstrong specificity has been found. Therefore, to seek tumor markers with highsensitivity and strong specificity to diagnose the colon cancer is still one of themedical researchers’objectives.The proteome refers to the whole protein expressed under specific time,environment and test condition. In the oncogenesis and development of the tumor,many proteins are over-active or under-active. It is of great significance to screenand evaluate the tumor markers in early diagnosis, early treatment and prognosis. Theproteomic technology, with the advantages of high throughput and high efficiency, iscapable of detecting all the proteins in the tissue. Through analysis of the differentialproteomics, people can discover the tumor marker with high sensitivity and strongspecificity early in the outbreak, which is of practical significance in guiding the earlydiagnosis, early treatment and prognosis.Two-dimensional liquid chromatography and mass spectrometry were performed to evaluate the differentially expressed protein of the rectal cancer tissues andpara-carcinoma tissue. The result shows that among the39differential proteins,19shows up-regulated expression and20shows down-regulated expression, which isconsistent with the experimental results in early experiment. These protein promisesto be high-sensitive and high-specify tumor marker of the colorectal cancer, but itneeds further clinical verifications. This research intends to carry out clinicalverifications on the nucleophosmin,(NPM).The nucleophosmin(NPM) is positioned in5q35, with the function of molecularchaperones. It includes3sub-types: NPM1、NPM2and NPM3, among which NPM1if profoundly researched. NPM is a multifunctional protein capable of rapidlyshuttling between cytoplasm and nucleus. It can also adjust proliferation andapoptosis of cells by means of all sorts of signal paths. Besides, it is able to getinvolved in controlling centrosome duplication and synthetic biology of the ribosomeas well as participating the generating of various tumors.In recent years, researches on NPM mainly concentrated on its relation with thehematological malignancy. Some literatures report that NPM have high expression inmany solid tumors, such as the liver cancer, gastric cancer, and prostate cancer.However, few researches are carried out on the developing of colorectal cancer and itsmechanism of action at home and abroad. Does specific receptor existed in and out ofthe cell of NPM protein? What is the signal transduction pathway to play a role? Howdoes it run in the development of the tumor? These problems all needs furtherresearches. Therefore, we intend to construct human NPM1prokaryotic expressionvector and express purified NPM1protein. By means of traditional hybridomatechnique, we also intend to make NPM1monoclonal antibody and apply it to detect the expression level of NPM1protein in tissues and tumor diagnose, targeted therapyand evaluation of the prognosis, which is of great theoretical and practical meaning indiscovering the function of NPM1protein and its role in the illness.The main results of the research are as follows:Part1: The proteomics researches on the difference between colorectalcancer and adjacent tissues by means of the two-dimensional liquidchromatography and mass spectrometryMethods:1. Clinical samples collection:18specimens of the cancer tissues andpara-carcinoma tissues respectively of the adenocarcinoma patients in DukesB.2. Protein concentrations were analyzed after the total protein of the abovespecimens were extracted.3. Preparing proteolysis poly-peptides mixtures of the specimens.4. Two-dimensional liquid chromatography and mass spectrometry analysis wereused to analyze the above poly-peptides mixtures, then database search with thespectrogram acquired was carried out.Results:1. Cancer tissue: related data of846proteins were obtained.2. Para-carcinoma tissue: related data of535proteins were obtained.3. There are39differential expression protein identifications screened betweencolorectal cancer tissues and adjacent tissues.19differential proteins areoverexpression, while20differential proteins are down-expression.Conclusions:1. Data evaluation of the colorectal cancer and adjacent tissues by means of the two-dimensional liquid chromatography and mass spectrometry were donesuccessfully.2. Through data evaluation and comparative analysis, we find that there are39differential expression protein identifications screened between colorectal cancertissues and adjacent tissues. Among them,19differential proteins are overexpression,while20differential proteins are down-expression.Part2: NPM1gene cloning, prokaryotic vector construction and fusionprotein expression and purifyingMethods:1. The sequences of gene encoding the NPM1Protein were analyzed after thegene was exrracted from Genbank, then the primers were designed to amplify thesequence encoding the NPM1Protein by PCR2. Recycled and purified PCR products were cloned into pET28a vector, thenrecombinant plasmids were transformed into DH5cell. The Positive clones withrecombinant construct were identified by PCR and confirmed by DNA sequencing.3. The sequenced recombinant plasmid pET28a:: NPM1was transformed intoBL21cells4. The Eseherichia coli BL21transformed with the recombinant PlasmidpET28a::NPM1was inoculated in the LB medium(including100g/ml Km) andinduced by IPTG at a concentration of1.0mM,37℃,250rpm for4.0hr.5. SDS-PAGE were used to identified the NPM1protein.6. The recombinant NPM1Protein was purified by Ni column chromatograph andSDS-PAGE electrophoresis respectively， then it was concentrated after Purified byelectroelution. The Purified recombinant protein was dialyzed in the PSB buffer solution at 4℃overnight, packed and stored at-80℃.Results:1. The human NPM1gene was synthesized and prokaryotic expression plasmidpET28a::NPM1was successfully constructed. Through DNA sequencing andNCBI-Blas comparison, the results were completely consistent with destinationsequence.2. Prokaryotic expression plasmid pET28a::NPM1was transformed into host toinduce protein expression. The purified protein was evaluated as interest protein bySDS-PAGE analysis.Conclusions:1. The prokaryotic expression plasmid pET28a::NPM1was successfullyconstructed.2. After induced by IPTG to the host, purified by Ni column chromatograph andelectroelution, concentrated by ultrafiltration, Highly purified NPM1protein wasobtained by.Part3: Preparation, purification and evaluation of NPM1monoclonalantibody.Methods:1. BALB/c mice were immuned by the immunogen emulsion made by Freund’sadjuvant and purified NPM1protein2. Using the classic cell fusion method, the spleen cells of the immunized mousewith the highest titer were fused with myeloma cells.3. Positive cell line producing monoclonal antibodies was identified bymeasuring the supernatant titer of the cell with ELISA 4. The screened positive cell lines were subcloned by using limiting dilution method.Part of the subcloned cell lines were used to detect the antibody subtype, and theremaining were cryopreserved.5. Ascites were prepared by mice injected with positive hybridoma cells, thenascites antibody titer was detected by ELISA6. NPM1monoclonal antibody was obtained after the ascites antibody proteinwas purified with affinity chromatography.7. Concentration, purity and titer of NPM1monoclonal antibody were detected,and NPM1monoclonal antibody was used to screen and identify the NPM1protein byImmunohistochemistry (IHC).Results:1. A cell line that is capable of stably secreting the anti-human NPM1monoclonal antibody was obtained.2. The subtype of NPM1monoclonal antibody is IgG1; antibody titer is1:720000; the antibody purity is over95%.Conclusions:1. A hybridoma cell line that is capable of stably secreting the anti-human NPM1monoclonal antibody was developed.2. Anti-human NPM1monoclonal antibody was prepared and identified.Part4: The relation of the expressions of NPM1in the colorectal cancerwith the clinicasl pathological parameter.Methods:1.105carcinoma tissues having a whole clinicopathologic data as the restinggroup and92adjacent normal tissues as the control group were respectively collected; the clinicopathologic data were grouped according to the reformed Dukesperiodization as well as the TNM staging methods raised and revised in the UnionInternational Contre LeCancer （UICC） and American Joint Committee onCancer(AJCC).2. NPM expressions of the carcinoma tissue and para-carcinoma tissue weredetected by means of the immunohisto chemistry technique and the relation of NPM1expression with the clinical pathological parameter of the colorectal cancer wereanalyzed via the statistics software SPSS.Results:1. When the mouse anti-recombinant human NPM1antibody were applied,positive expressions can be detected in the carcinoma tissue and para-carcinomatissue.2. In comparison with the adjacent normal tissue, the positive rate of the NPM1protein in the carcinoma tissue sample showed a upward trend.3. For carcinoma tissue sample with diameter of tumor higher than4.0cm, thepositive expression rate of the NPM1protein is higher than that of carcinoma tissuesample with diameter of tumor lower than4.0cm. In the Dukes period, the positiverate of the NPM1protein is prominently higher in period C and D than that in periodA and B; in the TNM staging of the colorectal cancer, the positive rate of the NPM1protein in the carcinoma tissue is obviously higher in the clinical stage III and IV thanthat in clinical stage I and II; the positive rate of the NPM1protein in carcinomatissue with lymph node metastasis is prominently higher than that without lymph nodemetastasis; the positive rate of the NPM1protein in carcinoma tissue with distantmetastasis is prominently higher than that without distant metastasis. Conclusions:1、The NPM1protein were up-regulated expression in the carcinoma tissue。2、The up-regulated expression of the NPM1protein is related with the size ofthe tumor, Dukes staging, TNM staging, lymphatic metastasis, distant metastasis.