1. Overview
Syphilis is a venereal disease with extremely complicated clinical manifestations that can almost involve all organs in the body. Although the ideal drug for the treatment of syphilis, penicillin, was discovered in the 40th century, this disease is still an important public health problem worldwide [1]. At present, the problem of artificial culture of Treponema pallidum (TP), the pathogen of syphilis, has not been solved, which has hindered the in-depth study of the pathogenic mechanism of TP, the development of vaccines, and the development of syphilis serological diagnostic technology. In the early 1980s, with the development of molecular biology technology, especially the emergence of genetic engineering (DNA recombination) technology, the study of Treponema pallidum entered a new stage [2]. At present, all genomic DNA sequences of TP have been resolved [ 1]. Through cloning of recombinant TP dNA and expression in E. coli, a variety of recombinant TP antigens were prepared, which provided a new way for the study of syphilis. Now review the research status of recombinant TP antigen in the past 10 years.
2. Characteristics and preparation of recombinant TP antigen
There are nearly 20 kinds of recombinant TP antigens, such as TpN15, TpN17, TpN19, TpN29 ～ 35 (TpD), TpN44.5 (TmP a), TpN47, TpN35 (Tmp c), etc. [3]. Now some of the recombinant TP antigens with application prospects are described below.
(1) TP15kD antigen [4]: ​​15kD protein is a lipoprotein. It was first isolated and identified by Hensel et al. In 1985. Its content in TP membrane protein is relatively small, but it is shown in immunoblotting experiments with human syphilis serum In addition, it has a strong hyperreactivity to the lymphocytes of experimental syphilis rabbits, indicating that the 15kD protein not only has strong humoral immunogenicity, but also has strong cell immunogenicity, but Centurion-Lara et al. [5] Experiments show that this protein does not have any immunoprotection against TP infection. The recombinant 15kD antigen gene was screened from the TP gene library with specific monoclonal antibodies by Norgard et al [4]. Its latest reported expression plasmid is pMALc2 [5], which contains a maltose binding protein gene upstream of the DNA insertion site, making the expressed fusion protein easy to isolate and purify.
(2) 4D antigen: 4D antigen is a TP outer membrane protein with a molecular weight of 19kD. Radolf et al [6] found that both the natural state and the recombinant 4D protein are cross-linked by disulfide bonds to form an oligomeric structure and are resistant to Proteases indicate that 4D antigen may play a very important role in maintaining the complete structure of the outer membrane of TP. The recombinant TP4D antigen was first expressed and purified by Feniger et al. In E coli RRI E. coli [7]. Borenstein et al. Used intramuscular injection and intravenous injection to explore the effect of recombinant TP 4D antigen on experimental plum blue, and showed that immunization of animals with 4D antigen can change the course of experimental syphilis. Have a partial protection effect. It is suggested that recombinant TP antigen is another possible way to prepare vaccines.
(3) TP24 kD antigen: This recombinant protein is a secreted protein expressed in E. coli with plasmid pUC8 as a vector [8], which has strong immunogenicity, and its DNA sequence encoding TP24kD antigen only exists in pathogenic This DNA sequence does not exist in the non-pathogenic TP gene, suggesting that the TP24kD antigen is related to the pathogenicity of TP, but the relationship with the pathogenicity of TP needs further study. The experiments of Hsu et al. Found that reproducing the expression of 24kD can change the growth morphology of E. coli, and the appearance of flat and rough colonies and filamentous growth different from that of general E. coli shows that the 24KD antigen is related to the growth characteristics of TP.
(4) TmP A and TmP B: TmP a is a TP membrane protein with a molecular weight of 42 kD. Schouls [9] et al. Constructed a plasmid vector pPLc245 that can express the recombinant protein in E. coli K-12 in large quantities. Ijsselmuiden et al [10] explored the significance of recombinant TmPA antigen in serological diagnosis by indirect enzyme-linked immunosorbent assay (ELISA) method, by conducting 148 untreated and 167 treated syphilis sera and 190 non-syphilis sera The sensitivity was: 76% for primary syphilis, 100% for second-stage syphilis, and 98% for early stable syphilis. The titers of anti-TmP a in syphilis patients within 1 year after treatment decreased significantly. It shows that TmP a is not only meaningful in the diagnosis of syphilis serum, but also expected to be used to monitor the therapeutic effect of syphilis. The TmP b gene is located downstream of the TmP A gene [9]. The experiments of Schouls et al. Showed that the anti-TmP b antibody transition trend decreased with the treatment of syphilis patients, so it is expected to be used for monitoring of syphilis treatment. However, Tmp b antigen alone is not suitable for serological diagnosis, because a large part of the serum of syphilis patients does not respond to Tmp B. Wicher et al. [11] immunized guinea pigs with recombinant TP antigen, and found that only TmP B in the recombinant TP antigen can change the course of syphilis, suggesting that recombinant TmP b antigen may have certain significance in vaccine preparation.
(5) TP47kD antigen: This protein is a TP-integrated membrane protein antigen. In 1986, Norgard et al. [12] first cloned and expressed recombinant 47kD antigen in E. coli. A multi-center study found [13] that this protein has strong immunogenicity, is abundant in TP, and is unique to pathogenic bacteria. Chamberlain et al. [12] used an expression vector containing T7-dependent RNA polymerase to establish a system for efficiently expressing recombinant 47kD, and biologically analyzed and compared this recombinant antigen with the natural 47kD protein isolated from TP. The results showed that this The 47kD antigens of the two sources have the same biological characteristics, and are protein-containing lipids, and their hydrophobicity may be related to pathogenicity. In order to explore the relationship between hydrophobic structure and antigenicity, Weigel et al. [14] expressed non-acylated hydrophilic recombinant 47kD antigen, and detected 116 cases of syphilis serum at different stages by immunoblotting test, indicating that the hydrophilic recombinant 47kD The antigenicity of the antigen remains unchanged, indicating that the acylation structure has nothing to do with the antigenicity.
In addition to the above protein antigens, there are a variety of TP recombinant antigens with strong antigenicity, such as Tromp2 (28kD) [15], TmPC (35kD) and 34kD antigens. The biological and immunological characteristics of these recombinant antigens need to be further explored.
3. Application of recombinant TP antigen in serological diagnosis
Although Fujimura et al. [16] tested the immunoreactivity of different recombinant antigens by ELISA, it was found that G17 is more reactive than M47, S42 and G15. However, in recent years, applied research tends to use two or more recombinant antigens in combination, synthesizing the different characteristics of two or more antigens, thereby making it more specific and sensitive.
(1) The combination of 15kD and 47kD [17]: 15kD and 47kD are currently considered to be the two most antigenic TP proteins, and Zrein et al.'S enzyme immune kit with recombinant 15kD and 47kD as antigens-ELISyph -The sensitivity and specificity of rG were evaluated. In the serum positive for TPHA test, the correlation between ELISyph-rG results and TPHA antibody titer was 0.94. After testing 1822 normal healthy volunteer sera negative for VDRL (440 of which were negative by TPHA), 4 positive sera were detected by ELISyph-rG method in these 1822 sera. Further confirmation of positive sera by FTA-ABS and WB methods showed that the sensitivity was 100% and the specificity was greater than 99.8%. It is believed that the ELISyph-rG method using recombinant 15 kD and 47 kD as antigens can replace VDRL and TPHA methods. Suitable for screening large samples.
(2) 15 kD, 17 kD, and 47 kD groups: These three TP antigens are stored throughout the course of syphilis [18], including late stage stable syphilis. Especially for syphilis patients with HIV infection, anti-47 kD antibodies can also be detected when the content of anti-TP is lower than that of general syphilis patients. Gerber et al [3] studied the combined application of three recombinant antigens. The results showed that of 18 patients with syphilis, 17 patients could detect antibodies throughout the course of the disease, while 42 patients had no antibody in the serum of normal people Positive, suggesting that this detection method has a high sensitivity and specificity. Young et al. [18] evaluated these three recombinant proteins as antigens and enzyme enzyme kits-ICE syphilis (immune-capture EIA), and compared with natural
The kit with TP as antigen——Captia SelectSyph-G, TPHA, FTA-ABS and VDRL were compared. The results showed that the specificity of ICE Syphilis was significantly higher than that of Captia selectSyph-G (99.8% and 99.2% respectively, P <0.02 > Its sensitivity is also significantly higher than Captia selectSyph-G, FTA-ABS and TPHA (99% to 100%, respectively, P <0.01, 91.4% to 92.4%, 92.4% and 97.1%>. In 15 cases with HIV Among the infected syphilis patients, the serum ICE syphilis test results were all positive, and the Captia selectSyph-G test results were negative in 3 cases. It can be seen that the high specificity and high sensitivity of the ICE syphilis test method will provide screening tests for syphilis An ideal means.
Young et al. also established a latex agglutination method for rapid detection of syphilis using these three recombinant proteins as antigens-Syphilis fast [19], and by testing 1518 random serum samples, the specificity of Captia selectSyph-G and VDRL was carried out. Comparison, and its sensitivity was evaluated in 99 syphilis sera of each stage and 15 sera positive for screening, its specificity (99.8%) was significantly higher than Captia SelectSyyph-G (99.2%) and VDRL (99.1%) ) (P <0.02>, but there is no significant difference between its sensitivity and Captia selectSyph-G (93% and 92.1%, respectively), but the sensitivity of these two methods are significantly higher than VDRL (46.5%) (P <0.001) It can be seen that Syphilis fast method is expected to become a syphilis screening method with high specificity, fast detection speed and easy operation.
4. Outlook
Farley et al. [20] believe that screening syphilis for the relevant population is one of the important measures to control and prevent syphilis. At present, there are two main serological tests routinely used for syphilis screening and diagnosis, one is the non-spiral test method for lipid antigens (such as VDRL, RPR), and the other is a specific Treponema pallidum test (such as TPHA, FTA-ABS). The former has low sensitivity and specificity, and there are false positives and false negatives, while the latter's biggest problem is the lack of antigen sources, which has not yet been solved. Because TP cannot be cultured artificially at present, the TP used for diagnostic tests is mainly obtained by inoculating rabbit testis, which requires certain laboratory conditions, and is time-consuming, laborious, and material-intensive. Therefore, to explore the application of recombinant TP antigen in the serological diagnosis of syphilis is one of the main purposes of preparing recombinant antigens. The preparation of recombinant proteins allows us to obtain different target antigens in the laboratory. This aspect is to establish the existing serological diagnosis of syphilis The alternative method provides the conditions, and at the same time lays the foundation for the establishment of new diagnostic methods with higher specificity and sensitivity, such as the preparation of monoclonal antibodies through recombinant antigens to establish a method for detecting syphilis antigens, which will Laboratory diagnosis of sexual syphilis provides new means. At the same time, through the analysis of the structure of different recombinant antigens, as well as biological and immunological analysis, the purpose of screening TP pathogenic factors and developing TP vaccines can be achieved, providing a more effective means for the prevention and treatment of TP.
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