AREQUIPA – PERÚ AÑO
3. PLANTEAMIENTO DEL PROBLEMA
enzymes. The anti-E3G Vj^ DNA and the Fv lys myc vectoi> without the anti-lysozyme were purified from the gel and ligated. The ligated vector was transformed in E, coli JM109 and colonies were picked and grown as for the gene ligation. Because of the internal Pst I site a digest with this enzyme would show successful ligations, as only vectors with the anti-E3G would give a band of around 600 bp (figure 2.6). This shows that most of the vectors contain anti-E3G DNA insert. Number 5 was chosen as the cleanest digest and used in the making the final construct. To avoid the problem of the the X ho I and the Pst I internal restriction sites the final construct was prepared by removing both myc genes by a Sac I and Eco RI digest, and the appropriate parts purified
from the gel: E3G lys myc pUC 19 (-V^ lys myc) and P^G myc.
These were ligated and the ligated vector transformed. Again a Pst I restriction enzyme digest would show a successful ligation, (figure 2.7). As each of the three different pUC 19 vectors were made, the colony which gave the best results was regrown and clone purified then made into glycerol slants. This allows more of any of these plasmids to be grown and purified when necessary. The final construct was purified in large amounts and a large part of this was further purified using PEG precipitation. This purified DNA was used in several sequence reactions using an automated DNA sequencer - again carried out by Dean Sibthorpe. The sequence for the two cloned variable genes are shown in table 2.2 and 2.3.
1 2 3 4 5 7 8 9 10 11 12
Figure 2.6 S a c l / X h o l restriction digest ofV% lys E3G myc pUC 19 showing clones with a positive ligation of light chain DNA.
1093 805 514 468
Figure 2.7S a d / X h o l restriction digest of Fv E3G myc pUC 19 showing clones with a positive ligation of light chain DNA.
base no. a.a. no.
GAC GTG GAG GTG AGG GAA AGT GGA GGG TGG - 3 0
D I E L T Q T P P S - 1 0
CTG GGT GTG AGT GTT GGA GAT GAG GTT TGG - 60
L P V S L G D Q V S - 2 0
ATG TGT TGG AGA TGT AGT GAG AGG GTT GTG - 9 0
I S G R S S Q S L V - 3 0
TGG AAT AAT AGA AGG AAG TAT TTA GAT TGG - 1 2 0
S N N R R N Y L H W — 4 0
TAG GTG GAG AAG GGA GGG GAG TGT GGA AAG - 1 5 0
Y L Q K P G Q S P K - 5 0
GTG GTG ATG TAG AAA GTT TGG AAG GGA TTT - 1 8 0
L V I Y K V S N R F - 6 0
TGT GGG GTG GGA GAG AGG TTG AGT GGG AGT - 2 1 0
S G V P D R F S G S - 7 0
GGA TGA GGG AGA GAT TTG AGA GTG AAG ATG - 2 4 0
G S G T D F T L K I - 8 0
AGG AGA GTG GGG GGT GAG GAT GTG GGA GTT - 2 7 0
S R V A A E D L G L - 9 0
TAT TTG TGG TGT GAA AGT TGA GAT GTT GGG - 3 0 0
Y F G S Q S S H V P - 1 0 0
GTG AGG TTG GGT TGT GGG ACC AAG GTG GAG - 3 3 0
L T F G S G T K L E - 1 1 0
ATG GAA CAA AAA CTC ATC TCA GAA GAG GAT - 3 6 0
I E Q K L I S E E D - 1 2 0
CTG AAT TAA L N
Table 2.2 DNA and derived protein sequence of anti-E3G antibody light chain variable gene witii myc tail. Hie myc tail DNA and amino acid sequences are shown in italics.
base no. a.a. no.
CAG GTG GAG GTG GAG GAG TGT GGG GGT GGG - 3 0
Q V Q L Q E S G G G - 1 0
TTG GTG AAG GTT GGA GGG TGT ATG AGT GTG - 60
L V N L G G S M T L - 2 0
TCG TGT GTA GGG TGT GGA TTG AGT TTG AAT - 90
S G V A S G F T F N - 3 0
AGG TAT TAG ATG TGT TGG GTT GGG CAG AGT - 1 2 0
T Y Y M S W V R Q T - 4 0
GGA GAG AAG AGG GTG GAG TTG GTG GGA GGG - 1 5 0
P E K T L E L V A A - 5 0
ATT AAT AGT GAT GGT GAA GGT ATG TAT TAT - 1 8 0
I N S D G E P I Y Y - 6 0
GGA AGA GAG TTG AAG GGG GGA GTG AGG ATG - 2 1 0
P D T L K G R V T I - 7 0
TGT GGA GAG AAT GGG AAG AAG AGG GTA TAG - 2 4 0
S R D N A K K T L Y - 8 0
GTG GAA ATG AGG AGT GTG AAG TTT GAG GAG - 2 7 0
L Q M S S L N F E D - 9 0
AGA GGG TTA TAT TAG TGT GGA AGA GTT AAT - 3 0 0
T A L Y Y G A R L N - 1 0 0
TAG GGG GTG TAT GGT ATG GAG TAT TGG GGG - 3 3 0
Y A V Y G M D Y W G - 1 1 0
GAA GGG AGG AGG GTG AGG GTG TGG TGA TAA
Q G T T V T V S S
Table 2.3 DNA and derived protein sequence of anti-E3G antibody heavy chain variable gene.
2.4 Conclusions.
Although the heavy chain variable gene was cloned without difficulty, the presence of rogue light chain variable genes in hybridoma cells, and an initial poor selection of 5' primers made cloning of the anti E3G
gene difficult. With the likelihood of pulling out rogue sequences, the use
efficient m ethod of cloning light chain variable genes: firstly by
multiplying the probability of obtaining the right gene; and secondly, by
allowing rapid screening of these products by negating the need to place
all these genes into vectors. The use of a large variety of primers also gives every chance possible of by-passing the 'rogue' sequence problem. Internal restriction endonuclease sites in both the and genes
required a slightly convoluted process of constructing the final plasmid,
but was extremely useful in providing a rapid method of differentiating
DNA which had the correct insert present. This was especially important where there was a significant count on transformation plates for the
negative control ligation. This meant once DNA from each culture was
checked by the restriction enzyme digest, that this DNA and the culture
itself could be used in subsequent reactions with confidence.
Once the genes are isolated there is a large scope of use for these genes to
be placed in other plasmids, for example; several plasmids exist with
different polypeptide tails, which can be of use in ELISA detection and also in purification. The variable genes could also be placed into a vector
which contains the DNA which codes for a polypeptide linker between the C-terminus of the Vg and the N-terminus of the V^. This would
create a scFv which can provide a more stable fragment that is possible to