| CEA was one of the first tumour-associated antigens to be identified and has been well characterised. CEA is an oncofetal glycoprotein,which is found at high levels in the fetal colon and at lower levels in the normal adult colonic epithelium. CEA occurs at abnormally high levels in several benign disorders and in some malignant tumours. The statistics displayed CEA in data enunciation large intestine expression rate as 83.3%, the cancer of the liver was 80%, lung cancer was76.6%, mammary glands cancer was 63.2%. The innate character of CEA was glycoprotein with complicated construction,180 kDa in molecular weight. CEA confirmed by the immunity fluorescence technique existed on the tumor cell membrane, was the construction glycoprotein of the cell membrane, and had in the expression on the cell membrane rate different. The technical of hybridoma made CEA- monoclonal antibody being used for biology research , medical science examinations and tumors' curing extensively. Human anti- mouse respond ( HAMA) and the complete monoclonal antibody that was big and hard entered the tumor organization limited its development. Ab phage display technology has been widely applied in recent years to make the human's antibody gene , the antibody bank and monoclonal antibody of human in germs, passing that technique particularly to imitate the antibody born process outside body without bying immunity system ,and displayed the captivating foreground.At setting up antibody library,There had many factors that influenced the quantity,the diversity and mature degrees of the antibody library and the size of the library capacity. Therefore, (1) the specimen was drew from the lymphoid knot in mesenterium. The lymph node was proprietary the important immune organ of the mammalian. The 95% cell inside the lymphoid follicle was B lymphocyte, and was big B lymphocyte of the conversion mostly. B lymphocytes were subjected to the choice function of the antigen that the follicle dendrite cell surface comes together, having been several splited and mutated round the process with membrane antibody construction, only its the antibody of membrane and surface antigens contain height affinity cell then can reserve to continue to split up and divide, rest then were all eliminated, so the high affinity antibody would be obtained. (2) Specimen source in the node, rectum cancer of the high expression CEA(>10 ng/ ml), to make the cell in the antibody mRNA with high and plentiful degree. (3) While withdrawing the total RNA, emphasize the RNA integrity, increase to withdraw and turn the diverse that the RNA step will reduce theRNA purely, affect the library to permit, so the RNA a value that attain OD260/ OD280 is above1. (4) Set up the light chain library first, the cloned heavy chain genes connects with each other, guaranteeing diversity of the heavy chain gene. (5) Makes the competence cell of having the high quantity, the strict electricity conversion operates, making a conversion rate attained 109, then can guarantee that the library permits to attain more than 107.To construct human phage antibody library Example of human phage antibody library obtained from lymphonode of ten patients with colon carcinoma. Concentrations of plasma CEA of every patient was more than 10 ng/ml. Employing routing RT-PCR with sets of oligonucleotide primers designed byΚang.The specific products were isolated by agar gel electrophoresis and purified with QIAquick Gel Extraction Kit.The purifiedκlight chain products were digested with SacⅠand Xba, then recombined with the phagemid p3MH. Product of ligation was transformed into E.coli XL1-Blue by electrotransformation.Aphage antibody library ofκlight chain containing 1.4×107members was constructed.The insert frequency of ligation was 70% determined by digesting DNA extracted from 10 clones.The phagemid p3MH-κof the library ofκlight chain was then extracted and recombined with the purified Fd gene product digested with SpeⅠand XhoⅠ.We constructed a phage antibody library of Fab whose capacity had had 5.2×106 members.The recombinant frequency of the Fab was 30% determined by digesting DNA extracted from 10 clones with SacⅠand SpeⅠ.In order to get a specific antibody to CEA from this antibody library with low Fab recombinant frequency, we take up a new panning strategy that is reducing the quantity of the coated antigen and increaseing washing time gradually during the fifth round panning. We use the human CEA as the coated antigen and use the new strategy to pan the antibody library. Five rounds of panning produced an 30 fold amplification in eluted phage, indicating enrichment for specific antigen-binding clones. After the fifth round of specific panning we got six phage antibodies to the human CEA protein. We constructed the expression phagemids and transformed E.coli XL1-blue to express soluble antibody, three of those expressed the soluble antibody successfully. Soluble Fab was expressed both in the bacterial culture supernatant and cell lysis. The supernatant and cell lysis showed clear reactivity to CEA antigen but not react with BSA or HBsAg by ELISA. Western blotting using the Rabbit anti human IgG(H+L)-HRP demonstrate the size of protein is about 48KD. Clone 4 did not show any reactivity with coated protein that show they were not expressed. DNA fragment coding for VH and Vκfrom the reactive clone 1 was sequenced .Results showed the VH gene segment was derived from VH3 gene family and VL gene segment from the VK gene family by INTERNET GenBank comparison.The success of isolating human anti-CEA Fab prove the usefulness of phage display system inhuman antibody preparation. Human monoclonal antibodies to CEA maybe useful as molecular tools to study the pathogenesis of tumor expressing CEA, as well as for the diagnostic and therapeutic purposes.
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