There are limitations to polyclonal and monoclonal antibodies that can be found. Antibodies desired for special purposes, for example investigation of enzyme catalysis, could require antibodies of such selectivity that few are likely to be found by conventional methods. While animals may synthesize hundreds of antibodies against a large antigen, the number is limited by the size of the B cell population. Even somatic mutation can be imagined not to generate all desired structures in antigen-binding sites of antibodies. How could vast numbers of variant antibodies be generated for the study of antibody-antigen interactions?

One method for increasing the number of antibody specificities available for study would be to express a large library of light and heavy chains in bacteria. If these large libraries could be combined, then the same multiplicative increase in diversity as occurs in nature could also be available. Furthermore, expressing the proteins in bacteria simplifies altering specific amino acids or mutagenizing regions of the protein. Time and cost scales would also become much more reasonable if the work is done in bacteria.

How might one begin to express a wide collection of light or heavy chains in bacteria? We cannot simply take a collection of V region genes, for most in the genome are not connected to the D or J regions and no diversification has occurred at the splicing step. We need to clone after rearrangement has taken place, and it would be best to clone only genes that have rearranged. If, however, we clone from DNA, the intervening sequences still interrupt the genes. The best approach is to clone from the mRNA using reverse transcriptase to make DNA copies. Then the polymerase chain reaction can be used to amplify the DNA. Due to the high fraction of conserved amino acids in the variable and light chain regions, it is possible to design oligonucleotides that will specifically hybridize to regions of the genes. The oligonucleotides can also be made with restriction enzyme cleavage sites so that later the resulting DNA can be easily inserted into a cloning vector (Fig.1).

Fig1. Amplification of an mRNA sequence coding for an immunoglobulin gene. Reverse transcriptase is used at the first step to make DNA. Thereafter the thermoresistant Taq polymerase is used in normal PCR reactions. The sequences encoding the restriction enzyme cleavage sites do not hybridize to the template on the first cycle. Thereafter they do.

Cleverness in designing the cloning vector also is helpful. The incoming DNA must be expressed; therefore the plasmid must contain an active promoter. It should be possible to regulate this promoter in case the gene products are lethal to the cells. The lac promoter is a good one to use. Downstream from the promoter must be a good ribosome-binding site. Beyond this it is helpful to have a sequence that will direct the resulting protein to be secreted from the cells. Then, at last, comes the cloning site which is flanked by the two restriction enzyme cleavage sites. The same sites were engineered into the DNA that was amplified by PCR. Finally, a translation termination sequence is needed. Additional factors are that after generating libraries of light and heavy chains, combinations of the two must be formed. Resulting vectors must carry one light and one heavy chain with both being expressed under control of the lac promoter (Fig. 2).

Fig2. Design of vectors suitable for expression of light chain or heavy chain fragments. The combinatorial vector expresses both the light chain and the fragment of the heavy chain.

Screening of the recombined library for vectors carrying a light and heavy chain that bind a particular antigen must be easy. Techniques have been developed for screening large numbers of lambda plaques on plates, and thus the best vector for initial screening would be lambda phage rather than a plasmid. Later, once a desired recombinant has been found, the light and heavy chains should be transferred to a plasmid vector. Finally, rather than attempting to synthesize the entire antibody molecule, just the Fab’ fragment should be sufficient for many studies, and the probes for PCR amplification can be designed accordingly.

The technology described above has worked well and libraries have been made containing up to 10⁶ different light chains and 10⁶ different heavy chain fragments. These have been combined to yield libraries with truly gargantuan numbers of different antibody specificities.

اخر الاخبار

اخبار العتبة العباسية المقدسة

مواكب العزاء تستذكر تضحيات الإمام الحسين (عليه السلام) في ذكرى الأربعين

مجمع الشيخ الكليني ينظم سفرة باسم السيدة أم البنين (عليها السلام)

شعبة التوجيه الديني النسوي تحيي ذكرى شهادة الإمام الرضا (عليه السلام)

بدعم العتبة العباسية المقدسة.. المفارز التطوعية تقدم خدماتها الصحية للزائرين

المزيد

الآخبار الصحية

قد يؤدي إلى فشل القلب.. تحذير من مسكن آلام يستعمله الملايين

هل الجري يساعد على فقدان الوزن؟.. إليك ما يقوله الخبراء

ماذا يحدث لجسمك عند شرب عصير البطيخ بانتظام؟

علماء يكتشفون أن نقص عنصر غذائي "شائع" قد يسبب الزهايمر

المزيد

الآخبار التكنلوجية

المعادن الحيوية قد تنضب خلال 10 سنوات.. دراسة صادمة ؟

"إنستغرام" يستنسخ خاصية شائعة من منافسه "سناب شات"

رواد فضاء يغادرون محطة الفضاء الدولية بعد مهمة استمرت 5 أشهر

"كريساليس".. سفينة فضائية ستسافر بالبشر إلى أعماق النجوم ؟!

المزيد

مواضيع ذات صلة

Heritable Genome Editing

Challenges to in Vivo Editing of Hematopoietic Stem Cells

EX Vivo Manufacturing of Therapeutic Cells

Using Engineered Nucleases to Initiate the Genome Editing Process

Base Editing

Double-stranded DNA breaks: the basis of nuclease-based genome editing

Plaque and Colony Hybridization for Clone Identification

Cloning from RNA

Plasmid Vectors

كلونة النعجة دولي Cloning Dolly the sheep

ادخال الجينات في الخلايا البدائية النواة

أدوات نقل الـ DNA نواقل الكلونة (cloning vehicles)