Record Details

Heterologous Expression of Pneumococcal Surface Antigen PsaA Gene

KrishiKosh

View Archive Info
 
 
Field Value
 
Title Heterologous Expression of Pneumococcal Surface Antigen PsaA Gene
 
Creator PATEL, NAFISABANU A.
 
Contributor MAHATMA, LALIT
 
Subject proteins, diseases, bacteria, biological phenomena, genes, amino acids, sampling, dna, enzymes, vegetables
 
Description Attempts were made to isolate Streptococcus pneumoniae from
the clinical samples. The clinical samples procurred from different
laboratories included sputum, blood, throat swab, bronchial and
tracheal aspirate. Typical cocci shaped organisms occurring in pair ar e
character ised, Gram positive reaction, alpha haemolysis, inability to
produce effervescence of CO2 in catalase test, susceptibility to
optochin and bile solublization were the basic tests performed for the
identification and characterization of S. pneumoniae. By the basic tests
and VITEK test, three isolates AML-4, AML-7 and ADL-1 wer e
identifiedand char acter ized to be S. pneumoniaewhere as the other
three alpha haemolytic streptococci ( ADL-2, AML-3 AND AML-10)
were identified as S. viridian and the other three isolates showieg beta
haemolysis were of other streptococci groups. S. pneumoniae
synthesize at least four different antigens produced by the surface of
the bacteria commonly known as Pneumococcal surface antigens (Psa)
which includes PsaA, PsaB, PsaC and PsaD. Bacter ia also have surface
proteins known as Pneumococal surface proteins. The resu lts of Pairwise
sequence alignment indicates PsaA gene is highly conserved as
compared to the PspA gene. The amino acid composition of PsaA
protein contained Maximum content of lysine in the range of 40
residues, while the PspA protein co ntained 90 residues of Alanine and
87 residues of Glutamine. PspA protein had large amount of negativel y
charged residues (121 aminoacids) while the PsaA protein had just 49
such r esidues. The estimated half life of both the proteins in
reticulocytes (in-vitro) was approximately 30 hours, in yeast it was
greater than 20 hours and in E. coli it was more than 10 hours. The
result of stabilit y analysis indicated that PsaA protein had 72.61%
stabilit y which was higher than the stabilit y index of PspA (58.19%
stabilit y). Major hydrophobicit y region of PsaA protein was between
the aminoacid positions 170-180. While the PspA protein graph
interpreted the hydrophobic region cover ing a larger part of the protein
associated between the regions 110-120 and then beginnign from the
aminoacid 180 till 600 aminoacids. Out of the 26 MHC ligand s
predicted for PspA protein, 22 epitopic sites lies between 200-630
aminoacids scoring PsaA protein as more stable than the PspA protein.
The number of MHC ligands identified in PsaA protein was 7, lying at
amino acid positions 61,78,79,118,202,266 and 290. No epitope had the
mutable site for PsaA protein. Looking at the results of bioinformatics
it was concluded that the PsaA gene is better choice for the
immuno genicit y. hydrophobicit y, maximum score of bulkiness and
mutabilit y score indicated that the PsaA gene is better choice for the
immuno genicit y. DNA could be extracted by the CTAB method with
slight modification. In PCR the temperature gradient of higher
temperature from 46ºC and above did not result into amplification of
PsaA gene. The best amplification was observed at 45.4 ºC. 1.0 mM
concentration of MgCl2 was found best for the specific amplification of
PsaA gene. The concentration of PCR product in the range of 50ng
resulted into positive ligation. The overnight incubation of the ligation
mixture at 4ºC incr eased the rate of positive ligation reaction. DH5α
stain of E.coli was used for transformation of PsaA gene.
Transformation of E.coli was performed on the plates containing
Ampicillin and X-gal and kept for incubation. The ratio of 1:3 of
vector to the PsaA gene resulted into increased transformation
efficiency. The r esults of controlled plate and transformation with
sample 1 and sample 2 showed the presence of transformed colonies
and successful cloning of PsaA gene into E.coli. Single digestion wit h
Kpn1 linear ized the plasmid giving a band of 4 kb. Crude protein was
extracted from the transformed cells and analysed for the expression of
PsaA protein. SDS-PAGE showed the presence of bands of PsaA
proteins of of 40 kDa indicating heterologous expressio n of PsaA in
E.coli.
For the effective sterilization treatment of leaves for 4 minutes
with the mercuric chloride was found most appropriate. For in vitro
regeneration of tomato, basal MS media fortified with 1.25 mg/l BAP+
0.50 mg/l IAA showed the best results in terms of callus induction
(86.67 %), days taken for the callus initiation (8.33 days), days taken
for shoot initiation ( 26 days of inoculation) and the number of shoots
formed (7.67). For shoot regeneration and multiplication 2 mg/l BAP +
1mg/l IAA) gave the highest response in terms of 61.11%, and the
number of shoots produced were 11 as well as the highest shoot length
of 5.17 cm. Subculturing effectively leads to increase in the number as
well as the length of shoots. The third subculture showed an increase in
the number of shoots regenerated to be nearly 12 as well as increase in
the shoot length to 5.90 cm. Rooting parameters were significantl y
influenced by the types and concentrations of Auxin and the strength of
MS medium. Half str ength MS medium supplemented with 0.1 mg/l
NAA (T4) recorded significantly minimum days to initiate the root (7
days), maximum rooting per cent (70 %) and number of root also
higher. The combination of half MS with the hormone concentr ation of
0.50 mg/l IBA also gave better rooting. During the hardening
maximum survival (61 %) of plantlets with minimum days for new
sprouting (11.25 days) was reported in Vermicompost : soil mixture.
Since the gene is highly conservative, this would be available in
all the serot ypes and strains of the pathogen. High stabilit y,
availab ilit y of all the epitopes on the surface, high antigenicity, long
retention time in Prokaryotes and Eukaryotes system further support
the hypothesis that it would not be digested by the phagocytes and will
be exposed to carry out the stimulation of immune system and all the
immuno genic sites will be exposed to the lymphocytes for the
production of systemic and specific antibodies against the prevailing as
well as newly emerging strains and serotypes of S. pneumoniae. The
expression of PsaA gene in the tomato and its comsumption would
elicit the immune system and stimulate the humoral immune response
against Streptococcus pneumoniae. This approach would help in the
prevention and eradication of highly fatal disease of human decreasing
the child mortalit y and increasing the life expectanc y.
 
Date 2016-05-04T11:19:20Z
2016-05-04T11:19:20Z
2013-07
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/65778
 
Language en
 
Format application/pdf
 
Publisher Navsari Agricultural University, Navsari