Use of recombinant CFP-10 protein for a skin test specific for Mycobacterium tuberculosis infection

Xueqiong Wu*, Shisheng Feng, Haiqing Duan, Lingxia Zhang, Junxian Zhang, Yourong Yang,
Yan Liang and Yingchang Shi

According to the World Health Organization, Mycobacterium tuberculosis has infected approximately one thirt of the world population and more than 8 million new cases of tuberculosis worldwide was reported in 2002. It is estimated that 45% of Chines population are latently infected with MTB, among which 10% latently infected individuals may become activated later in their lives. Proper identification of latently infected individuals for prophylactic chemotherapy is needed to prevent future reactivation of MTB infection and to control TB epidemics in China. At present, the purified protein derivative PPD  also known as the tuberculin skin test based on the delayed type hypersensitivity DTH reaction is widely used to detect MTB infection. However, the specificity of PPD skin test is low, because the antigens in PPD are common to many mycobacterial species including Mycobacterium bovis Bacills Calmette Guerin BCG, the vaccine strain being used widely for immunization against MTB infection. As a result the PPD skin test cannot be used to definitively identify MTB infection.

Mycobacterial antigens that can distinguish the MTB specific DTH reaction from that induced by the BCG vaccination are highly desirable and their utility in skin test specific for TB diagnosis needs be demonstrated.

The genetic region of difference 1- RD1 is present in the genomes of MTB and M.bovis but it is absent in all strains of M.bovis BCG, as well as most non tuberculosis mycobacteria NTM including Mycobacterium avium and Mycobacterium intracellulare. Both CFP-10 and ESAT-6 antigens are encoded by Rv3874 and Rv3875 genes, respectively, in one operon within the RD1 region and are expressed simultaneously at a similar ration. Because of their absence in BCG and NTM strains, ESAT-6 and CFP-10 have been extensively investigated as MTB specific antigens and shown to have great potential as the specific antigens for the diagnosis of MTB infection in humans. The immune responses found in experimental animals and humans to the recombinant CFP-10 or ESAT-6 proteins are similar to those found for the CFP-10 or ESAT-6 peptides. However, there were differences in immune responses of human or cattle to individual CFP-10 and ESAT-6 antigens, determined by ELISPOT assay, 18% people tested were positive for CFP-10 peptides, 30% were positive for ESAT-6 peptides, 33% were positive for both peptides and 34% were positive for the ESAT-6/CFP-10 fusion protein; 39% subjects had either a positive result for a peptide or a positive result for the fusion protein. The difference may be explained by polymorphism in the human leucocyte antigen HLA type in the population and difference of MTB stains infected in different countries. However, the use of a single antigen in the skin test may exclude those who do not have memory T cells specific of rESAT-6 even after exposure to MTB due to their unique HLA type. In this study, we evaluated the utility of rCFP-10 protein as a stimulating antigen to improve the sensitivity of rESAT-6 skin test or to find a better antigen that ESAT-6 for differential diagnosis of MTB infection.

MATERIALS AND METHODS

Bacterial Strains and Products

Escherichia coli strain BL21(DE3) was grown in Luria Bertani-LB liquid and solid media Mycobacterium reference stains including MTB(H37Rv), M.bovis, and M.bovis BCG Danish were used. Stock culture of MTB, M.bovis and M.bovis BCG Danish were grown on Lowenstein-Jensen slants at 37 degree C for 4 weeks and then trasferred to Sauton liquid medium at 37 degree C without shaking for 4 weeks. PPD produced from MTB(50 IU/ml) was used.

Gene Cloning, expression and protein purification

The MTB CFP-10 cloning, expression and purification were performed with standard procedures described by Sambrook et al. Briefly, the gene encoding the MTB CFP-10 protein was amplified by PCR using two primers. The forward primer contained a kpn1 restriction enzyme recognition site: -

5’-CGAGATCTGGGTACCGACGACGACGACAAGATGGCAGAGATGAAGACCGA-3’.  The reverse primer contained an EcoR1 restriction enzyme recognition site:-

5’-CGGAATTCT CAGAAGCCCATTTGCGAG G-3’.

PCR amplification is performed by 30 cycles of denaturation at 94 degree C for 30s, primer annealing at 55 degree C for 30s, extension at 68 degree C for 1 min and final extension at 72 degree C for 2 min. PCR products were digested by the restrictive enzyme Kpn1 and EcoR1. The resulting fragments were ligated with T4 DNA ligase into a Kpn-1 and EcoR-1 digested pET-32a plasmid vector containing the kanamycin resistant gene. The recombinant plasmids were transferred into E.coli BL21(DE3) and kanamycin resistant transformants were selected on LB agar plate containing kanamycin (50 microgram/ ml) and 5-bromo-4-choro-3-indolyl Beta D-galactopyranoside (X-Gal) (20 microgram/ml). The plasmid from one kanamycin resistant transformant was sequenced , using the same primers as for PCR amplification. Sequences were compared with that registered in the GenBank database by Basic Local Alignment Search Tool BLAST analysis.

The expression of rCFP-10 as an N-termnal 6xhistidine-tagged protein was induced by 1.0mM Isopropyl-b-D-thiogalactopyranoside(IPTG), in solube form after incubation at 30 degree C for 4 h. Soluble rCFP-10 was purified by metal chelate affinity chromotography under the native condition, the fusion tag was then cleaved with enterokinase and the resulting tag-free rCFP-10 was further purified by affinity chromatography according to the purification procedure provided by the expression vector manufacturer.

The freshly prepared rCFP-10 protein was suspended in 0.1mM phophate buffered saline(PBS, pH=7.0) and the solution was filtered by passing it through the Acrodisc Syringe Filter (0.45 micro meter) with low protein binding. The protein concentration was determined spectrophotometrically. The final protein solution was aliquoted into small glass ampules, each contained 1 mg of purified rCFP-10 protein. The sample were then lyphilized sealed and stored at –20 degree C. The protein was reconstituted in 1 ml saline before use.

Determination of molecular mass and purity

The molecular mass of purified rCFP-10 protein was determined by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular masses of the protein standards ranged from 6 to 97.4 kDa. Gels were stained with Coomassie blue. The molecular mass and purity of rCFP-10 protein were analyzed and calculated with TotalLab v1.11. The molecular mass of purified rCFP-10 protein was further confirmed by mass spectrometry.

Guinea pig sensitization and skin tests

38 NIH white guinea pig weighing 250 to 300 g were used and maintained under specific pathogen free conditions. There was a female to male ration of 1:1 . In the first experiment, 8 guinea pig(4 male and 4 femaile) were sensitized by hypodermic injection of 5 mg autoclaved MTB in 0.1ml of saline in the groin once weekly for four weeks. In the second experiment, 6 guinea pigs were sensitized by peritoneal injection of 100 live MTB in 0.5 ml saline and the skin test on these animals was performed 5 weeks after senitization. In the third experiment, 24 guinea pigs were divided into 3 groups and sensitized by hypodermic injection of 0.1 ml live BCG-Danish Vaccine(group 1), 5 mg autoclaved MTB (group2) or 5 mg autoclaved M.bovis (group 3) in animal’s groin area once weekly for four weeks.

For skin testing, guinea pigs were shaved on the back and injected intradermally with 0.8 to 1.2 microgram of the purified rCFP-10 protein in 0.1 ml of PBS or 0.1 ml (5 lU) of PPD or 1.0 microgram of the purified rESAT-6 protein  0.1 ml of PBS as a control for four to eight weeks following sensitization. The diameters of both axes of skin erythema were independently measured and recorded at 24,48 and 72 h after antigen injection. Results were expressed as means of diameter in millimeters of erythema.

RESULTS

Cloning, expression and purification of CFP-10

The nucleotide sequence encoding the rCFP-10 had 100% homologous identity with CFP-10 sequence reported in GenBank database. The molecular mass obtained from rCFP-10 fusion protein as determined by SDS-PAGE was 30.8 kDa. In order to determine an ideal condition for rCFP-10 protein expression, we compared the levels of expression under various IPTG concentrations and different induction time and temperatures. The results showed that IPTG concentrations had no effect on the level of the expression, whereas the induction time and temperature affected the expression level significantly. The amount of rCFP-10 protein that existed in soluble form was at 35% to 50% of the total soluble proteins when the induction took place under 1.0 mM IPTG at 30 degree C for 4 h. The purified rCFP-10 protein showed only one band when analyzed by 15% SDS PAGE with a purity of more than 90%. The molecular mass of the purified rCFP-10 protein was 14.8 kDa as determined by SDS-PAGE and 13.8 kDa by mass spectrometry.

DTH reactivity to rCFP-10 in guinea pigs sensitized with killed MTB

Different doses of rCFP-10 protein were tested for their ability to produce DTH responses in guinea pigs sensitized with killed MTB. In the first study, each of the seven guinea pigs(1 female was dead during the immunization) was injected intradermally at five sites with PBS as the negative control, PPD (5 lU) as the positive control and 3 doses of rCFP-10(0.8, 1.0 and 1.2 microgram). All three doses of rCFP-10 antigen were able to induce positive skin reaction at a similar level as compared to the positive control PPD at 24 and 48 h after injection as defined by an erythema response greater than 5 mm in diameter. The difference in skin reaction was  not statistically significant. The positive responses measured at the 24 h time point were generally stronger than that at 48h for all groups, though there was no statistically significant difference. By 72 hrs, the reaction to PPD had decreased markedly as compared to the reactions at the previous time points. All reaction to rCFP-10 reduced to less than 5 mm in diameter and were therefore considered negative.

DISCUSSION

It has been shown that ESAT-6 could be used as an antigen in skin test for the detection of MTB infection. However, the population with different HLA type may recognize different epitopes. CFP-10 may induce DTH responses in people who do not respond to ESAT-6, overcoming the problems related to genetic restriction in antigen recognition and improving the sensitivity of skin test. Thus the ability of CFP-10 to elicit DTH responsive in experimental animals sensitized with various strains of MTB complex should be evaluated to prepare a new antigen of skin test.

 

African Journal of Biotechnology Vol. 9(42), pp. 7180-7185, 18 October, 2010
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 ©2010 Academic Journals